Compositions comprising bacterial strains

ABSTRACT

The invention provides compositions comprising bacterial strains for treating and preventing inflammatory and autoimmune diseases.

CROSS-REFERENCE

This application is a continuation of U.S. application Ser. No.16/248,857, filed Jan. 16, 2019, now U.S. Pat. No. 10,864,236, issuedDec. 15, 2020, which is a continuation of U.S. application Ser. No.15/700,007, filed on Sep. 8, 2017, now U.S. Pat. No. 10,744,167, issuedAug. 18, 2020, which is a continuation of International Application No.PCT/GB2016/051776, filed Jun. 15, 2016, which claims the benefit ofGreat Britain Application No. 1510467.2, filed Jun. 15, 2015; and GreatBritain Application No. 1520501.6, filed Nov. 20, 2015; all of which arehereby incorporated by reference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Aug. 26, 2020, isnamed 56708_717_304_sequence_listing.txt and is 2,887,608 bytes in size.

TECHNICAL FIELD

This invention is in the field of compositions comprising bacterialstrains isolated from the mammalian digestive tract and the use of suchcompositions in the treatment of disease.

BACKGROUND TO THE INVENTION

The human intestine is thought to be sterile in utero, but it is exposedto a large variety of maternal and environmental microbes immediatelyafter birth. Thereafter, a dynamic period of microbial colonization andsuccession occurs, which is influenced by factors such as delivery mode,environment, diet and host genotype, all of which impact upon thecomposition of the gut microbiota, particularly during early life.Subsequently, the microbiota stabilizes and becomes adult-like [1]. Thehuman gut microbiota contains more than 500-1000 different phylotypesbelonging essentially to two major bacterial divisions, theBacteroidetes and the Firmicutes [2]. The successful symbioticrelationships arising from bacterial colonization of the human gut haveyielded a wide variety of metabolic, structural, protective and otherbeneficial functions. The enhanced metabolic activities of the colonizedgut ensure that otherwise indigestible dietary components are degradedwith release of by-products providing an important nutrient source forthe host. Similarly, the immunological importance of the gut microbiotais well-recognized and is exemplified in germfree animals which have animpaired immune system that is functionally reconstituted following theintroduction of commensal bacteria [3-5].

Dramatic changes in microbiota composition have been documented ingastrointestinal disorders such as inflammatory bowel disease (IBD). Forexample, the levels of Clostridium cluster XIVa bacteria are reduced inIBD patients whilst numbers of E. coli are increased, suggesting a shiftin the balance of symbionts and pathobionts within the gut [6-9].Interestingly, this microbial dysbiosis is also associated withimbalances in T effector cell populations.

In recognition of the potential positive effect that certain bacterialstrains may have on the animal gut, various strains have been proposedfor use in the treatment of various diseases (see, for example,[10-13]). Also, certain strains, including mostly Lactobacillus andBifidobacterium strains, have been proposed for use in treating variousinflammatory and autoimmune diseases that are not directly linked to theintestines (see [14] and [15] for reviews). However, the relationshipbetween different diseases and different bacterial strains, and theprecise effects of particular bacterial strains on the gut and at asystemic level and on any particular types of diseases, are poorlycharacterised.

There is a requirement in the art for new methods of treatinginflammatory and autoimmune diseases. There is also a requirement forthe potential effects of gut bacteria to be characterised so that newtherapies using gut bacteria can be developed.

SUMMARY OF THE INVENTION

The inventors have developed new therapies for treating and preventinginflammatory and autoimmune diseases. In particular, the inventors havedeveloped new therapies for treating and preventing diseases andconditions mediated by IL-17 or the Th17 pathway. In particular, theinventors have identified a new bacterial strain that is effective forreducing the Th17 inflammatory response. As described in the examples,oral administration of compositions comprising the bacterium depositedunder accession number NCIMB 42380 may reduce the severity of theinflammatory response, including the Th17 inflammatory response, inmouse models of asthma, rheumatoid arthritis and multiple sclerosis. Asalso described in the examples, oral administration of compositionscomprising the bacterium deposited under accession number NCIMB 42380may reduce tumor size in mouse models of cancer that may be associatedwith the Th17 inflammatory response.

Therefore, in a first embodiment, the invention provides a compositioncomprising the bacterium deposited under accession number NCIMB 42380 ora biotype thereof, for use in a method of treating or preventing adisease or condition mediated by IL-17 or the Th17 pathway. Theinventors have identified that treatment with such bacterial strains canreduce levels of cytokines that are part of the Th17 pathway, includingIL-17, can alleviate the Th17 inflammatory response and can provideclinical benefits in mouse models of inflammatory and autoimmunediseases mediated by IL-17 and the Th17 pathway.

In particular embodiments, the invention provides a compositioncomprising the bacterium deposited under accession number NCIMB 42380 ora biotype thereof, for use in a method of treating or preventing adisease or condition selected from the group consisting of multiplesclerosis; arthritis, such as rheumatoid arthritis, osteoarthritis,psoriatic arthritis, or juvenile idiopathic arthritis; neuromyelitisoptica (Devic's disease); ankylosing spondylitis; spondyloarthritis;psoriasis; systemic lupus erythematosus; inflammatory bowel disease,such as Crohn's disease or ulcerative colitis; celiac disease; asthma,such as allergic asthma or neutrophilic asthma; chronic obstructivepulmonary disease (COPD); cancer, such as breast cancer, colon cancer,lung cancer or ovarian cancer; uveitis; scleritis; vasculitis; Behcet'sdisease; atherosclerosis; atopic dermatitis; emphysema; periodontitis;allergic rhinitis; and allograft rejection. The effect shown for thebacterium deposited under accession number NCIMB 42380 on the Th17inflammatory response may provide therapeutic benefits for diseases andconditions mediated by IL-17 and the Th17 pathway, such as those listedabove.

In preferred embodiments, the invention provides a compositioncomprising the bacterium deposited under accession number NCIMB 42380 ora biotype thereof, for use in a method of treating or preventing asthma,such as neutrophilic asthma or allergic asthma. The inventors haveidentified that treatment with the bacterium deposited under accessionnumber NCIMB 42380 can reduce recruitment of neutrophils and eosinophilsinto the lungs, which can help treat or prevent asthma. Furthermore, theinventors have tested and demonstrated the efficacy of the bacteriumdeposited under accession number NCIMB 42380 in mouse models of asthma.In certain embodiments, the composition is for use in a method oftreating or preventing neutrophilic asthma or eosinophilic asthma. Theeffect shown for the compositions of the invention on neutrophils andeosinophils mean that they may be particularly effective for treating orpreventing neutrophilic asthma and eosinophilic asthma. Indeed, incertain embodiments, the composition is for use in a method of reducinga neutrophilic inflammatory response in the treatment or prevention ofasthma, or the composition is for use in a method of reducing aneosinophilic inflammatory response in the treatment or prevention ofasthma. In certain embodiments, the invention provides a compositioncomprising the bacterium deposited under accession number NCIMB 42380 ora biotype thereof, for use in the treatment of asthma. In especiallypreferred embodiments, the invention provides a composition comprisingthe bacterium deposited under accession number NCIMB 42380, for use inthe treatment of asthma, and in particular neutrophilic asthma. Thebacterium deposited under accession number NCIMB 42380 is shown to havea particularly pronounced effect on neutrophils in asthma models andtreatment with this bacterium may be particularly effective for treatingneutrophilic asthma.

In further preferred embodiments, the invention provides a compositioncomprising the bacterium deposited under accession number NCIMB 42380 ora biotype thereof, for use in a method of treating or preventingrheumatoid arthritis. The inventors have identified that treatment withthe bacterium deposited under accession number NCMB 42380 can provideclinical benefits in a mouse model of rheumatoid arthritis and canreduce joint swelling. In preferred embodiments, the invention providesa composition comprising the bacterium deposited under accession numberNCIMB 42380 or a biotype thereof, for use in the treatment of rheumatoidarthritis. Compositions using the bacterium deposited under accessionnumber NCIMB 42380 may be particularly effective for treating rheumatoidarthritis.

In further preferred embodiments, the invention provides a compositioncomprising the bacterium deposited under accession number NCIMB 42380 ora biotype thereof, for use in a method of treating or preventingmultiple sclerosis. The inventors have identified that treatment withthe bacterium deposited under accession number NCMB 42380 can reducedisease incidence and disease severity in a mouse model of multiplesclerosis. In preferred embodiments, the invention provides acomposition comprising the bacterium deposited under accession numberNCIMB 42380 or a biotype thereof, for use in the treatment of multiplesclerosis. Compositions using the bacterium deposited under accessionnumber NCIMB 42380 may be particularly effective for treating multiplesclerosis.

In further preferred embodiments, the invention provides a compositioncomprising the bacterium deposited under accession number NCIMB 42380 ora biotype thereof, for use in a method of treating or preventing cancer,such as breast, lung or liver cancer. The inventors have identified thattreatment with the bacterium deposited under accession number NCIB 42380can reduce tumour growth in mouse models of breast, lung and livercancer. In certain embodiments, the composition is for use in a methodof reducing tumour size or preventing tumour growth in the treatment ofcancer.

In further preferred embodiments, the invention provides a compositioncomprising the bacterium deposited under accession number NCIMB 42380 ora biotype thereof, for use in a method of treating or preventinguveitis, such as posterior uveitis. Compositions comprising thebacterium deposited under accession number NCIMB 42380 or a biotypethereof may be particularly effective for treating uveitis.

In certain embodiments, the compositions of the invention are for use ina method of reducing IL-17 production or reducing Th17 celldifferentiation in the treatment or prevention of a disease or conditionmediated by IL-17 or the Th17 pathway. In particular, the compositionsof the invention may be used in reducing IL-17 production or reducingTh17 cell differentiation in the treatment or prevention of asthma,rheumatoid arthritis or multiple sclerosis or of asthma, rheumatoidarthritis, multiple sclerosis, cancer or uveitis. Preferably, theinvention provides compositions comprising the bacterium deposited underaccession number NCIMB 42380 or a biotype thereof for use in reducingIL-17 production or reducing Th17 cell differentiation in the treatmentor prevention of asthma, rheumatoid arthritis or multiple sclerosis. Theinvention also provides compositions comprising the bacterium depositedunder accession number NCIMB 42380 or a biotype thereof for use inreducing IL-17 production or reducing Th17 cell differentiation in thetreatment or prevention of cancer.

In certain embodiments, the composition is for use in a patient withelevated IL-17 levels or Th17 cells. The effect on the Th17 inflammatoryresponse shown for the bacterium deposited under accession number NCIMB42380 may be particularly beneficial for such patients.

In preferred embodiments of the invention, the bacterial strain in thecomposition is the bacterium deposited under accession number NCIMB42380. Biotype bacterial strains may also be used, such as bacterialstrains that have a 16s rRNA sequence that is at least 95%, 96%, 97%,98%, 99%, 99.5% or 99.9% identical to the 16s rRNA sequence of thebacterium deposited under accession number NCIMB 42380. Preferably, thebacterial strain has a 16s rRNA sequence that is at least 95%, 96%, 97%,98%, 99%, 99.5% or 99.9% identical to SEQ ID NO:1. Preferably, thebacterial strain for use in the invention has the 16s rRNA sequencerepresented by SEQ ID NO:1.

In certain embodiments, the composition of the invention is for oraladministration. Oral administration of the strains of the invention canbe effective for treating IL-17- or Th17 pathway-mediated diseases andconditions. Also, oral administration is convenient for patients andpractitioners and allows delivery to and/or partial or totalcolonisation of the intestine.

In certain embodiments, the composition of the invention comprises oneor more pharmaceutically acceptable excipients or carriers.

In certain embodiments, the composition of the invention comprises abacterial strain that has been lyophilised. Lyophilisation is aneffective and convenient technique for preparing stable compositionsthat allow delivery of bacteria.

In certain embodiments, the invention provides a food product comprisingthe composition as described above.

In certain embodiments, the invention provides a vaccine compositioncomprising the composition as described above.

Additionally, the invention provides a method of treating or preventinga disease or condition mediated by IL-17 or the Th17 pathway, comprisingadministering a composition comprising the bacterium deposited underaccession number NCIMB 42380 or a biotype thereof.

In developing the above invention, the inventors have identified andcharacterised a bacterial strain that is particularly useful fortherapy. The bacterium deposited under accession number NCIMB 42380 isshown to be effective for treating the diseases described herein, suchas arthritis, asthma and multiple sclerosis. Therefore, in anotheraspect, the invention provides a cell of the bacterium deposited underaccession number NCIMB 42380, or a derivative thereof. The inventionalso provides compositions comprising such cells, or biologically purecultures of such cells. The invention also provides a cell of thebacterium deposited under accession number NCIMB 42380, or a derivativethereof, for use in therapy, in particular for the diseases describedherein. The bacterium deposited under accession number NCIMB 42380 isalso shown to be effective for treating cancer.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1: Mouse model of house dust mite-induced asthma—Total BAL fluidcell counts.

FIG. 2: Mouse model of house dust mite-induced asthma—Total eosinophilcount in BALF.

FIG. 3: Mouse model of house dust mite-induced asthma—Proportion ofeosinophils in BALF.

FIG. 4: Mouse model of house dust mite-induced asthma—Total macrophagecount in BALF.

FIG. 5: Mouse model of house dust mite-induced asthma—Proportion ofmacrophages in BALF.

FIG. 6: Mouse model of house dust mite-induced asthma—Total neutrophilcount in BALF.

FIG. 7: Mouse model of house dust mite-induced asthma—Proportion ofneutrophils in BALF.

FIG. 8: Mouse model of house dust mite-induced asthma—Total lymphocytecount in BALF.

FIG. 9: Mouse model of house dust mite-induced asthma—Proportion oflymphocytes in BALF.

FIG. 10: Mouse model of severe neutrophilic asthma—Total BAL fluid cellcounts.

FIG. 11: Mouse model of severe neutrophilic asthma—Total eosinophilcount in BALF.

FIG. 12: Mouse model of severe neutrophilic asthma—Proportion ofeosinophils in BALF.

FIG. 13: Mouse model of severe neutrophilic asthma—Total macrophagecount in BALF.

FIG. 14: Mouse model of severe neutrophilic asthma—Proportion ofmacrophages in BALF.

FIG. 15: Mouse model of severe neutrophilic asthma—Total neutrophilcount in BALF.

FIG. 16: Mouse model of severe neutrophilic asthma—Proportion ofneutrophils in BALF.

FIG. 17: Mouse model of severe neutrophilic asthma—Total lymphocytecount in BALF.

FIG. 18: Mouse model of severe neutrophilic asthma—Proportion oflymphocytes in BALF.

FIG. 19: Mouse model of rheumatoid arthritis—Bodyweights, days −14 to 0.Data are presented as Mean±SEM percentages of the initial (Day −14)bodyweights. Statistical significance: ▴p<0.05 and ▴▴▴▴p<0.0001 whencompared to the vehicle-treated group.

FIG. 20: Mouse model of rheumatoid arthritis—Bodyweights, days 0 to 42.Data are presented as Mean±SEM percentages of the initial (Day 0)bodyweights. ▴p<0.05, ♦p<0.05, ▴▴▴p<0.001, ••••p<0.0001 when compared tothe vehicle-treated group.

FIG. 21: Mouse model of rheumatoid arthritis—Clinical Scores. Data arepresented as Mean±SEM. ****p<0.0001 when compared to Day 21 in thevehicle-treated group. ♦p<0.05 when compared to the vehicle-treatedgroup on a given day.

FIG. 22: Mouse model of rheumatoid arthritis—Splenocyte proliferativeresponse to Collagen II. Media background subtracted[CII-stimulated—media background] counts per minute based on 3H-TdRincorporation. All data are presented as Mean±SEM. **p<0.01 compared toVehicle group.

FIG. 23: Mouse model of rheumatoid arthritis—Levels of IFNγ in tissueculture supernatants. Lines represent group median values.

FIG. 24: Mouse model of rheumatoid arthritis—Levels of IL-17A in tissueculture supernatants. Lines represent group median values.

FIG. 25: Mouse model of rheumatoid arthritis—Levels of IL-10 in tissueculture supernatants. Lines represent group median values.

FIG. 26: Mouse model of rheumatoid arthritis—Levels of IL-6 in tissueculture supernatants. Lines represent group median values.

FIG. 27: Histopathology scoring system.

FIG. 28: Mouse model of house dust mite-induced asthma—Total IgE inSerum

FIG. 29: Mouse model of house dust mite-induced asthma—HDM specific IgG1in Serum

FIG. 30: Mouse model of house dust mite-induced asthma—Total IgE in BALF

FIG. 31: Mouse model of house dust mite-induced asthma—HDM specific IgG1in BALF

FIG. 32: Mouse model of house dust mite-induced asthma—HistologicalAnalysis—Mean Peribronchiolar Infiltration Score

FIG. 33: Mouse model of house dust mite-induced asthma—HistologicalAnalysis—Mean Perivascular Infiltration Score

FIG. 34: Mouse model of house dust mite-induced asthma—HistologicalAnalysis—Mean Inflammatory Score (Average of both Peribronchiolar andPerivascular Infiltration Score)

FIG. 35: Mouse model of house dust mite-induced asthma—HistologicalAnalysis—Mucus Score

FIG. 36: Mouse model of house dust mite-induced asthma—IL-9 level inlung tissue

FIG. 37: Mouse model of house dust mite-induced asthma—IL-1a level inlung tissue

FIG. 38: Mouse model of house dust mite-induced asthma—IFNg level inlung tissue

FIG. 39: Mouse model of house dust mite-induced asthma—IL-17A level inlung tissue

FIG. 40: Mouse model of house dust mite-induced asthma—IL-4 level inlung tissue

FIG. 41: Mouse model of house dust mite-induced asthma—IL-5 level inlung tissue

FIG. 42: Mouse model of house dust mite-induced asthma—IL-1b level inlung tissue

FIG. 43: Mouse model of house dust mite-induced asthma—RANTES level inlung tissue

FIG. 44: Mouse model of house dust mite-induced asthma—MIP-1a level inlung tissue

FIG. 45: Mouse model of house dust mite-induced asthma—KC level in lungtissue

FIG. 46: Mouse model of house dust mite-induced asthma—MIP-2 level inlung tissue

FIG. 47: Mouse model of severe neutrophilic asthma—HDM specific IgG1 inSerum

FIG. 48: Mouse model of severe neutrophilic asthma—HDM specific IgG2a inSerum

FIG. 49: Mouse model of severe neutrophilic asthma—HDM specific IgG1 inBALF

FIG. 50: Mouse model of severe neutrophilic asthma—HDM specific IgG2a inBALF

FIG. 51: Mouse model of severe neutrophilic asthma—HistologicalAnalysis—Mean Peribronchiolar Infiltration Score

FIG. 52: Mouse model of severe neutrophilic asthma—HistologicalAnalysis—Mean Perivascular Infiltration Score

FIG. 53: Mouse model of severe neutrophilic asthma—HistologicalAnalysis—Mean Inflammatory Score (Average of both Peribronchiolar andPerivascular Infiltration Score)

FIG. 54: Mouse model of severe neutrophilic asthma—TNFa level in lungtissue

FIG. 55: Mouse model of severe neutrophilic asthma—IL-1a level in lungtissue

FIG. 56: Mouse model of severe neutrophilic asthma—IFNg level in lungtissue

FIG. 57: Mouse model of severe neutrophilic asthma—IL-17F level in lungtissue

FIG. 58: Mouse model of severe neutrophilic asthma—IL-1b level in lungtissue

FIG. 59: Mouse model of severe neutrophilic asthma—RANTES level in lungtissue

FIG. 60: Mouse model of severe neutrophilic asthma—MIP-2 level in lungtissue

FIG. 61: Mouse model of severe neutrophilic asthma—KC level in lungtissue

FIG. 62: Mouse model of severe neutrophilic asthma—IL-17A level in lungtissue

FIG. 63: Mouse model of severe neutrophilic asthma—MIP-1a level in lungtissue

FIG. 64: Mouse model of severe neutrophilic asthma—IL-33 level in lungtissue

FIG. 65: Mouse model of rheumatoid arthritis—Visual Template forHistopathology Scoring. Representative images showing composite scoresfrom mouse tarsal joints in a collagen-induced arthritis study.

FIG. 66: Mouse model of rheumatoid arthritis—Histopathology:Inflammation Scores. Data are presented as Mean±SEM. **p<0.01 whencompared to the vehicle-treated group.

FIG. 67: Mouse model of rheumatoid arthritis—Histopathology: CartilageScores. Data are presented as Mean±SEM. ***p<0.001 when compared to thevehicle-treated group.

FIG. 68: Mouse model of rheumatoid arthritis—Histopathology: BoneScores. Data are presented as Mean±SEM. ***p<0.001 when compared to thevehicle-treated group.

FIG. 69: Mouse model of rheumatoid arthritis—Histopathology: TotalScores. Data are presented as Mean±SEM. *p<0.05, ***p<0.001 whencompared to the vehicle-treated group.

FIG. 70: Mouse model of rheumatoid arthritis—Histopathology:Representative Pictures. Animal ID (#n.n) and limb (R for right, L forleft) are indicated between brackets. Top left image (vehicle):extensive joint and bone destruction with inflammation and fibrosisextending to the peri-articular soft tissues.

FIG. 71: Mouse model of multiple sclerosis—clinical score.

FIG. 72: Mouse model of multiple sclerosis—disease incidence.

FIG. 73: Mouse model of breast cancer—tumor volume.

FIG. 74: Mouse model of lung cancer—tumour volume.

FIG. 75: Mouse model of liver cancer—liver weight.

FIG. 76: Attachment of MRX004 and B. breve type strains to human cells.

FIG. 77: Exopolysaccharide production assay.

FIG. 78: Bound and released exopolysaccharide production by MRX004.

FIG. 79: Attachment of MRX004 to Caco-2 cells.

FIG. 80: Rapid ID 32 A profile of MRX004 alone (A) and in comparisonwith B. breve type strains (B). White=negative reaction (no colourchange), Downwards cross-hatched=intermediate positive reaction (weakcolour change) and Black=positive reaction (strong appropriate colourchange).

FIG. 81: API® 50 CH analysis of MRX004. Upward cross-hatched=negativereaction (no colour change), Downward cross-hatch=intermediate positivereaction (weak colour change), Black=positive reaction (strongappropriate colour change) and White=doubtful reaction (unexpectedcolour change).

DISCLOSURE OF THE INVENTION

Bacterial Strains

The compositions of the invention comprise the bacterium deposited underaccession number NCIMB 42380 or a biotype thereof. The examplesdemonstrate that such bacteria are useful for treating or preventingdiseases and conditions mediated by IL-17 or the Th17 pathway. Theexamples also demonstrate that such bacteria are useful for treating orpreventing cancer. The preferred bacterial strain is the bacteriumdeposited under accession number NCIMB 42380.

The bacterium deposited under accession number NCIMB 42380 was tested inthe Examples and is also referred to herein as strain 751 or MRX004. Apartial 16S rRNA sequence for the 751 strain that was tested is providedin SEQ ID NO:1. Strain 751 was deposited with the internationaldepositary authority NCIMB, Ltd. (Ferguson Building, Aberdeen, AB21 9YA,Scotland) by GT Biologics Ltd. (Life Sciences Innovation Building,Aberdeen, AB25 2ZS, Scotland) on 12 Mar. 2015 and was assigned accessionnumber NCIMB 42380. GT Biologics Ltd. subsequently changed its name to4D Pharma Research Limited.

All microorganism deposits were made under the terms of the BudapestTreaty and thus viability of the deposit is assured. Maintenance of aviable culture is assured for 30 years from the date of deposit. Duringthe pendency of the application, access to the deposit will be affordedto one determined by the Commissioner of the United States Patent andTrademark Office to be entitled thereto. All restrictions on theavailability to the public of the deposited microorganisms will beirrevocably removed upon the granting of a patent for this application.The deposit will be maintained for a term of at least thirty (30) yearsfrom the date of the deposit or for the enforceable life of the patentor for a period of at least five (5) years after the most recent requestfor the furnishing of a sample of the deposited material, whichever islongest. The deposit will be replaced should it become necessary due toinviability, contamination or loss of capability to function in themanner described in the specification.

A genome sequence for strain 751 is provided in SEQ ID NO:2.

Bacterial strains that are biotypes of the bacterium deposited underaccession number NCIMB 42380 are also expected to be effective fortreating or preventing diseases and conditions mediated by IL-17 or theTh17 pathway. Bacterial strains that are biotypes of the bacteriumdeposited under accession number NCIMB 42380 are also expected to beeffective for treating or preventing cancer. A biotype is a closelyrelated strain that has the same or very similar physiological andbiochemical characteristics.

In certain embodiments, the bacterial strain for use in the inventionhas a 16s rRNA sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.5%or 99.9% identical to the 16s rRNA sequence of the bacterium depositedunder accession number NCIMB 42380. Preferably, the bacterial strain foruse in the invention has a 16s rRNA sequence that is at least 95%, 96%,97%, 98%, 99%, 99.5% or 99.9% identical to SEQ ID NO:1. Preferably, thebacterial strain for use in the invention has the 16s rRNA sequencerepresented by SEQ ID NO:1.

Alternatively, strains that are biotypes of the bacterium depositedunder accession number NCIMB 42380 and that are suitable for use in theinvention may be identified by sequencing other nucleotide sequences forthe bacterium deposited under accession number NCIMB 42380. For examplesubstantially the whole genome may be sequenced and a biotype strain foruse in the invention may have at least 95%, 96%, 97%, 98%, 99%, 99.5% or99.9% sequence identity across at least 80% of its whole genome (e.g.across at least 85%, 90%, 95% or 99%, or across its whole genome). Othersuitable sequences for use in identifying biotype strains may includehsp60 or repetitive sequences such as BOX, ERIC, (GTG)s, or REP or [16].Biotype strains may have sequences with at least 95%, 96%, 97%, 98%,99%, 99.5% or 99.9% sequence identity to the corresponding sequence ofthe bacterium deposited under accession number NCIMB 42380.

In certain embodiments, the bacterial strain for use in the inventionhas a genome with sequence identity to SEQ ID NO:2. In preferredembodiments, the bacterial strain for use in the invention has a genomewith at least 90% sequence identity (e.g. at least 92%, 94%, 95%, 96%,97%, 98%, 99% or 100% sequence identity) to SEQ ID NO:2 across at least60% (e.g. at least 65%, 70%, 75%, 80%, 85%, 95%, 96%, 97%, 98%, 99% or100%) of SEQ ID NO:2. For example, the bacterial strain for use in theinvention may have a genome with at least 90% sequence identity to SEQID NO:2 across 70% of SEQ ID NO:2, or at least 90% sequence identity toSEQ ID NO:2 across 80% of SEQ ID NO:2, or at least 90% sequence identityto SEQ ID NO:2 across 90% of SEQ ID NO:2, or at least 90% sequenceidentity to SEQ ID NO:2 across 100% of SEQ ID NO:2, or at least 95%sequence identity to SEQ ID NO:2 across 70% of SEQ ID NO:2, or at least95% sequence identity to SEQ ID NO:2 across 80% of SEQ ID NO:2, or atleast 95% sequence identity to SEQ ID NO:2 across 90% of SEQ ID NO:2, orat least 95% sequence identity to SEQ ID NO:2 across 100% of SEQ IDNO:2, or at least 98% sequence identity to SEQ ID NO:2 across 70% of SEQID NO:2, or at least 98% sequence identity to SEQ ID NO:2 across 80% ofSEQ ID NO:2, or at least 98% sequence identity to SEQ ID NO:2 across 90%of SEQ ID NO:2, or at least 98% sequence identity to SEQ ID NO:2 across100% of SEQ ID NO:2.

Alternatively, strains that are biotypes of the bacterium depositedunder accession number NCIMB 42380 and that are suitable for use in theinvention may be identified by using the accession number NCIMB 42380deposit and restriction fragment analysis and/or PCR analysis, forexample by using fluorescent amplified fragment length polymorphism(FAFLP) and repetitive DNA element (rep)-PCR fingerprinting, or proteinprofiling, or partial 16S or 23s rDNA sequencing. In preferredembodiments, such techniques may be used to identify strains of the samespecies as the bacterium deposited under accession number NCIMB 42380.

In certain embodiments, strains that are biotypes of the bacteriumdeposited under accession number NCIMB 42380 and that are suitable foruse in the invention are strains that provide the same pattern as thebacterium deposited under accession number NCIMB 42380 when analysed byamplified ribosomal DNA restriction analysis (ARDRA), for example whenusing Sau3AI restriction enzyme (for exemplary methods and guidance see,for example, [17]). Alternatively, biotype strains are identified asstrains that have the same carbohydrate fermentation patterns as thebacterium deposited under accession number NCIMB 42380.

Bacterial strains that are biotypes of the bacterium deposited underaccession number NCIMB 42380 and that are useful in the compositions andmethods of the invention may be identified using any appropriate methodor strategy, including the assays described in the examples. Forinstance, biotypes for use in the invention may be identified byculturing in anaerobic YCFA and/or administering the bacteria to thetype II collagen-induced arthritis mouse model and then assessingcytokine levels. In particular, bacterial strains that have similargrowth patterns, metabolic type and/or surface antigens to the bacteriumdeposited under accession number NCIMB 42380 may be useful in theinvention. A biotype strain will have comparable immune modulatoryactivity to the NCIMB 42380 strain. In particular, a biotype strain willelicit comparable effects on the asthma, arthritis, multiple sclerosisand cancer disease models and comparable effects on cytokine levels tothe effects shown in the Examples, which may be identified by using theculturing and administration protocols described in the Examples.

A particularly preferred strain of the invention is the bacteriumdeposited under accession number NCIMB 42380. This is the exemplary 751strain tested in the examples and shown to be effective for treatingdisease. Therefore, the invention provides a cell, such as an isolatedcell, of the bacterium deposited under accession number NCIMB 42380, ora derivative thereof. The invention also provides a compositioncomprising a cell of the bacterium deposited under accession numberNCIMB 42380, or a derivative thereof. The invention also provides abiologically pure culture of the bacterium deposited under accessionnumber NCIMB 42380. The invention also provides a cell of the bacteriumdeposited under accession number NCMB 42380, or a derivative thereof,for use in therapy, in particular for the diseases described herein.

A derivative of the bacterium deposited under accession number NCIMB42380 may be a daughter strain (progeny) or a strain cultured(subcloned) from the original. A derivative of a strain of the inventionmay be modified, for example at the genetic level, without ablating thebiological activity. In particular, a derivative strain of the inventionis therapeutically active. A derivative strain will have comparableimmune modulatory activity to the NCIMB 42380 strain. In particular, aderivative strain will elicit comparable effects on the asthma,arthritis, multiple sclerosis and cancer disease models and samecomparable effects on cytokine levels to the effects shown in theExamples, which may be identified by using the culturing andadministration protocols described in the Examples. A derivative of theNCIMB 42380 strain will generally be a biotype of the NCMB 42380 strain.

References to cells of the bacterium deposited under accession numberNCIMB 42380 encompass any cells that have the same safety andtherapeutic efficacy characteristics as the strain deposited underaccession number NCIMB 42380, and such cells are encompassed by theinvention.

In preferred embodiments, the bacterial strains in the compositions ofthe invention are viable and capable of partially or totally colonisingthe intestine.

In certain embodiments, the bacterial strain for use in the inventionhas low adherence to human intestinal epithelial cells, in particularCaco-2 cells. In a preferred embodiment, the bacterial strain for use inthe invention has low adherence to human intestinal epithelial cells, inparticular Caco-2 cells, in YCFA compared to Bifidobacteria, inparticular B. breve. In certain embodiments, the bacterial strain foruse in the invention exhibits adherence of less than 1% of totalculture, such as preferably less than 0.5% or less than 0.3%, whentested under the conditions described in Example 12.

In certain embodiments, the bacterial strain for use in the inventionproduces exopolysaccharides, for example wherein the exopolysaccharidesare bound to the extracellular surface of the bacterial strain. Incertain embodiments, the production of the bound exopolysaccharidesincreases the adhesion of the bacterial strain for use in the inventionto mucus or to the surface of epithelial cells, for example humanintestinal epithelial cells. In a preferred embodiment, the bacterialstrain for use in the invention produces more bound surfaceexopolysaccharides compared to Bifidobacteria, in particular B. breve.

In a preferred embodiment, the bacterial strain for use in the inventionboth has low adherence to human intestinal epithelial cells, inparticular Caco-2 cells, in YCFA compared to Bifidobacteria, inparticular B. breve (such as adherence of less than 1% of total culture,such as preferably less than 0.5% or less than 0.3%, when tested underthe conditions described in Example 12), and produces more bound surfaceexopolysaccharides compared to Bifidobacteria, in particular B. breve.

In certain preferred embodiments, the bacterial strain for use in theinvention is able to ferment the polysaccharide raffinose, for examplewhen cultured in an appropriate suspension medium (such as APIsuspension medium) at 37° C. for 4 hours.

In certain embodiments, the bacterial strain for use in the inventionhas reduced ability to ferment α-glucosidase and/or β-glucosidasecompared to Bifidobacteria, in particular B. breve, for example whencultured in an appropriate suspension medium (such as API suspensionmedium) at 37° C. for 4 hours.

In certain embodiments, the bacterial strain for use in the inventioncomprises one or more of the genes listed in Table 1, such as 5, 10, 20,50 or all of the genes in Table 1. In certain embodiments, the bacterialstrain for use in the invention comprises one or more of the geneslisted in Table 1 that are highlighted with single underlining, such asTransmembrane component BL0694 of energizing module of predicted ECFtransporter and/or Duplicated ATPase component BL0693 of energizingmodule of predicted ECF transporter. In certain embodiments, thebacterial strain for use in the invention comprises one or more of thegenes listed in Table 1 that are highlighted with double underlining andin bold, such as 1, 2, 3, 4 or 5 genes selected from: maltodextringlucosidase (EC 3.2.1.20), putative galactosidase, cellulose synthase(UDP-forming) (EC 2.4.1.12), chitinase (EC 3.2.1.14) and sensorybox/GGDEF family protein. In certain embodiments, the bacterial strainfor use in the invention comprises one or more of the genes listed inTable 1 that are highlighted with italics, such as 1, 2, 3, 4, 5, 6, 7,8 or 9 genes selected from: omega-3 polyunsaturated fatty acid synthasesubunit PfaA, Type I polyketide synthase, putative glycosyl hydrolase ofunknown function (DUF1680), ATPase component BioM of energizing moduleof biotin ECF transporter, Cation-transporting ATPase E1-E2 family,Ribose ABC transport system permease protein RbsC (TC 3.A.1.2.1), RiboseABC transport system ATP-binding protein RbsA (TC 3.A.1.2.1), 3′-to-5′oligoribonuclease (om), membrane protein related to Actinobacillusprotein (1944168).

In preferred embodiments, the bacterial strain for use in the inventioncomprises one or more (such as 5, 10, 15, 20, 25, 30, 40, 45, 50 or all)genes selected from:2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylic-acidsynthase (EC 2.2.1.9); 3′-to-5′ oligoribonuclease (om);Alpha-galactosidase (EC 3.2.1.22); ATPase component of generalenergizing module of ECF transporters; ATPase component STY3233 ofenergizing module of queuosine-regulated ECF transporter; ATP-dependentDNA helicase recG (EC 3.6.1.-); Beta-glucosidase (EC 3.2.1.21);Cellulose synthase (UDP-forming) (EC 2.4.1.12); Chitinase (EC 3.2.1.14);COG1309: Transcriptional regulator; D-alanyl-D-alanine carboxypeptidase(EC 3.4.16.4); Duplicated ATPase component BL0693 of energizing moduleof predicted ECF transporter; Fructokinase (EC 2.7.1.4);Glucose/mannose:H+ symporter GlcP; Glycosyltransferase (EC 2.4.1.-); GMPsynthase [glutamine-hydrolyzing] (EC 6.3.5.2); Hypothetical sugar kinasein cluster with indigoidine synthase indA, PkB family of kinases;Inosine-uridine preferring nucleoside hydrolase (EC 3.2.2.1); LSUribosomal protein L31 p @ LSU ribosomal protein L31 p, zinc-independent;LSU ribosomal protein L33p @ LSU ribosomal protein L33p,zinc-independent; Maltodextrin glucosidase (EC 3.2.1.20); Membraneprotein, related to Actinobacillus protein (1944168); Membrane-boundlytic murein transglycosylase D precursor (EC 3.2.1.-);Methyltransferase (EC 2.1.1.-); NADH-dependent butanol dehydrogenase A(EC 1.1.1.-); Phosphoglycolate phosphatase (EC 3.1.3.18);Phosphoribosylanthranilate isomerase (EC 5.3.1.24); Putative glycosylhydrolase of unknown function (DUF1680); Rhamnose-containingpolysacharide translocation permease; Ribokinase (EC 2.7.1.15); RiboseABC transport system, ATP-binding protein RbsA (TC 3.A.1.2.1); RiboseABC transport system, ATP-binding protein RbsA (TC 3.A.1.2.1); RiboseABC transport system, high affinity permease RbsD (TC 3.A.1.2.1); RiboseABC transport system, periplasmic ribose-binding protein RbsB (TC3.A.1.2.1); Ribose ABC transport system, permease protein RbsC (TC3.A.1.2.1); Ribose ABC transport system, permease protein RbsC (TC3.A.1.2.1); Sorbitol dehydrogenase (EC 1.1.1.14); SSU ribosomal proteinS14p (S29e) @ SSU ribosomal protein S14p (S29e), zinc-independent;Substrate-specific component STY3230 of queuosine-regulated ECFtransporter; Sucrose-6-phosphate hydrolase (EC 3.2.1.B3); Teichoic acidexport ATP-binding protein TagH (EC 3.6.3.40); Transmembrane componentBL0694 of energizing module of predicted ECF transporter; Transmembranecomponent STY3231 of energizing module of queuosine-regulated ECFtransporter; Two-component response regulator colocalized with HrtABtransporter; Type I restriction-modification system,DNA-methyltransferase subunit M (EC 2.1.1.72); Type Irestriction-modification system, restriction subunit R (EC 3.1.21.3);Type I restriction-modification system, specificity subunit S (EC3.1.21.3); Type I restriction-modification system, specificity subunit S(EC 3.1.21.3); Type I restriction-modification system, specificitysubunit S (EC 3.1.21.3); Xylitol dehydrogenase (EC 1.1.1.9); and XyloseABC transporter, periplasmic xylose-binding protein XylF. In preferredembodiments, the bacterial strain for use in the invention comprises oneor more (such as 5, 10, 15, 20, 25, 30, 35 or all) genes that are listedin the preceding sentence and that are not highlighted in Table 1.

Therapeutic Uses

As demonstrated in the examples, the bacterial compositions of theinvention are effective for reducing the Th17 inflammatory response. Inparticular, treatment with compositions of the invention achieves areduction in IL-17A levels and other Th17 pathway cytokines, andclinical improvements in animal models of conditions mediated by IL-17and the Th17 pathway. Therefore, the compositions of the invention maybe useful for treating or preventing inflammatory and autoimmunediseases, and in particular diseases or conditions mediated by IL-17. Inparticular, the compositions of the invention may be useful for reducingor preventing elevation of the IL-17 inflammatory response.

Th17 cells are a subset of T helper cells that produce, for example,IL-17A, IL17-F, IL-21 and IL-22. Th17 cell differentiation and IL-17expression may be driven by IL-23. These cytokines and others formimportant parts of the Th17 pathway, which is a well-establishedinflammatory signalling pathway that contributes to and underlies anumber of inflammatory and autoimmune diseases (as described in, forexample, [18-23]). Diseases wherein the Th17 pathway is activated areTh17 pathway-mediated diseases. Th17 pathway-mediated diseases can beameliorated or alleviated by repressing the Th17 pathway, which may bethrough a reduction in the differentiation of Th17 cells or a reductionin their activity or a reduction in the level of Th17 pathway cytokines.Diseases mediated by the Th17 pathway may be characterised by increasedlevels of cytokines produced by Th17 cells, such as IL-17A, IL-17F,IL-21, IL-22, IL-26, IL-9 (reviewed in [24]). Diseases mediated by theTh17 pathway may be characterised by increased expression ofTh-17-related genes, such as Stat3 or IL-23R. Diseases mediated by theTh17 pathway may be associated with increased levels of Th17 cells.

IL-17 is a pro-inflammatory cytokine that contributes to thepathogenesis of several inflammatory and autoimmune diseases andconditions. IL-17 as used herein may refer to any member of the IL-17family, including IL-17A, IL-17B, IL-17C, IL-17D, IL-17E, and IL-17F.IL-17-mediated diseases and conditions are characterised by highexpression of IL-17 and/or the accumulation or presence ofIL-17-positive cells in a tissue affected by the disease or condition.Similarly, IL-17-mediated diseases and conditions are diseases andconditions that are exacerbated by high IL-17 levels or an increase inIL-17 levels, and that are alleviated by low IL-17 levels or a reductionin IL-17 levels. The IL-17 inflammatory response may be local orsystemic.

Examples of diseases and conditions that may be mediated by IL-17 or theTh17 pathway include multiple sclerosis; arthritis, such as rheumatoidarthritis, osteoarthritis, psoriatic arthritis, or juvenile idiopathicarthritis; neuromyelitis optica (Devic's disease); ankylosingspondylitis; spondyloarthritis; psoriasis; systemic lupus erythematosus;inflammatory bowel disease, such as Crohn's disease or ulcerativecolitis; celiac disease; asthma, such as allergic asthma or neutrophilicasthma; chronic obstructive pulmonary disease (COPD); cancer, such asbreast cancer, colon cancer, lung cancer or ovarian cancer; uveitis;scleritis; vasculitis; Behcet's disease; atherosclerosis; atopicdermatitis; emphysema; periodontitis; allergic rhinitis; and allograftrejection. In preferred embodiments, the compositions of the inventionare used for treating or preventing one or more of these conditions ordiseases. In further preferred embodiments, these conditions or diseasesare mediated by IL-17 or the Th17 pathway.

In certain embodiments, the compositions of the invention are for use ina method of reducing IL-17 production or reducing Th17 celldifferentiation in the treatment or prevention of a disease or conditionmediated by IL-17 or the Th17 pathway. In certain embodiments, thecompositions of the invention are for use in treating or preventing aninflammatory or autoimmune disease, wherein said treatment or preventionis achieved by reducing or preventing elevation of the Th17 inflammatoryresponse. In certain embodiments, the compositions of the invention arefor use in treating a patient with an inflammatory or autoimmunedisease, wherein the patient has elevated IL-17 levels or elevated Th17cells or is exhibiting a Th17 inflammatory response. In certainembodiments, the patient may have been diagnosed with a chronicinflammatory or autoimmune disease or condition, or the composition ofthe invention may be for use in preventing an inflammatory or autoimmunedisease or condition developing into a chronic inflammatory orautoimmune disease or condition. In certain embodiments, the disease orcondition may not be responsive to treatment with TNF-α inhibitors.These uses of the invention may be applied to any of the specificdisease or conditions listed in the preceding paragraph.

IL-17 and the Th17 pathway are often associated with chronicinflammatory and autoimmune diseases, so the compositions of theinvention may be particularly useful for treating or preventing chronicdiseases or conditions as listed above. In certain embodiments, thecompositions are for use in patients with chronic disease. In certainembodiments, the compositions are for use in preventing the developmentof chronic disease.

The compositions of the invention may be useful for treating diseasesand conditions mediated by IL-17 or the Th17 pathway and for addressingthe Th17 inflammatory response, so the compositions of the invention maybe particularly useful for treating or preventing chronic disease,treating or preventing disease in patients that have not responded toother therapies (such as treatment with TNF-α inhibitors), and/ortreating or preventing the tissue damage and symptoms associated withIL-17 and Th17 cells. For example, IL-17 is known to activate matrixdestruction in cartilage and bone tissue and IL-17 has an inhibitoryeffect on matrix production in chondrocytes and osteoblasts, so thecompositions of the invention may be useful for treating or preventingbone erosion or cartilage damage.

In certain embodiments, treatment with compositions of the inventionprovides a reduction or prevents an elevation in IL-17 levels, inparticular IL-17A levels. In certain embodiments, treatment withcompositions of the invention provides a reduction or prevents anelevation in IFN-γ, IL-13, RANTES, MIP-1a, IL-8 or IL-6 levels. Suchreduction or prevention of elevated levels of these cytokines may beuseful for treating or preventing inflammatory and autoimmune diseasesand conditions, in particular those mediated by IL-17 or the Th17pathway.

In certain embodiments, treatment with the compositions of the inventionprovides a block of the attachment to or invasion of human cells, forexample human epithelial cells by pathogenic cells, for example E. coliand/or S. enteritidis.

In certain embodiments, treatment with the compositions of the inventionreduces or prevents the binding of pathogenic cells, for example E. coliand/or S. enteritidis, to the human epithelial cells, for example humanintestinal epithelial cells.

In certain embodiments, the production and release of exopolysaccharidesby the bacterial strains of the compositions of the invention may haveprotective effects against pathogenic species, for example E. coliand/or S. enteritidis. In certain embodiments, the production andrelease of exopolysaccharides by the bacterial strains of thecompositions of the invention may mediate the effect of the bacteria onthe IL-17 or Th17 pathway and may influence the host immune response. Incertain embodiments, the compositions of the invention are for use inproducing exopolysaccharides in the treatment of inflammatory andautoimmune diseases, and in particular diseases or conditions mediatedby IL-17.

In certain embodiments, the low adherence to human intestinal epithelialcells, in particular Caco-2 cells, of the bacterial strains of thecompositions of the invention may increase the beneficial effect of thecompositions of the invention on the IL-17 or the Th17 pathway and ondiseases mediated by IL-17 or the Th17 pathway.

In certain embodiments, treatment with compositions of the inventionprovides increased fermentation of raffinose in the intestine. Theexamples demonstrate that the bacterial strains of the compositions ofthe invention ferment the polysaccharide raffinose, and raffinosefermentation may confer effects on the host such as increased caecalbutyrate and increased gastrointestinal proliferation. In certainembodiments, the compositions of the invention are for use in increasingfermentation of raffinose in the intestine in the treatment ofinflammatory and autoimmune diseases, and in particular diseases orconditions mediated by IL-17.

Asthma

In preferred embodiments, the compositions of the invention are for usein treating or preventing asthma. The examples demonstrate that thecompositions of the invention achieve a reduction in the recruitment ofneutrophils and/or eosinophils into the airways following sensitisationand challenge with house dust mite extract and so they may be useful inthe treatment or prevention of asthma. Asthma is a chronic diseasecharacterised by inflammation and restriction of the airways. Theinflammation in asthma may be mediated by IL-17 and/or Th17 cells, andso the compositions of the invention may be particularly effective forpreventing or treating asthma. The inflammation in asthma may bemediated by eosinophils and/or neutrophils.

In certain embodiments, the asthma is eosinophilic or allergic asthma.Eosinophilic and allergic asthma are characterised by increased numbersof eosinophils in peripheral blood and in airway secretions and isassociated pathologically with thickening of the basement membrane zoneand pharmacologically by corticosteroid responsiveness [25].Compositions that reduce or inhibit eosinophil recruitment or activationmay be useful for treating or preventing eosinophilic and allergicasthma.

In additional embodiments, the compositions of the invention are for usein treating or preventing neutrophilic asthma (or non-eosinophilicasthma). High neutrophil numbers are associated with severe asthma thatmay be insensitive to corticosteroid treatment. Compositions that reduceor inhibit neutrophil recruitment or activation may be useful fortreating or preventing neutrophilic asthma.

Eosinophilic and neutrophilic asthma are not mutually exclusiveconditions and treatments that help address either the eosinophil andneutrophil responses may be useful for treating asthma in general.

Increased IL-17 levels and activation of the Th17 pathway are associatedwith severe asthma, so the compositions of the invention may be usefulfor preventing the development of severe asthma or for treating severeasthma.

In certain embodiments, the compositions of the invention are for use inmethods reducing an eosinophilic inflammatory response in the treatmentor prevention of asthma, or for use in methods of reducing aneutrophilic inflammatory response in the treatment or prevention ofasthma. As noted above, high levels of eosinophils in asthma isassociated pathologically with thickening of the basement membrane zone,so reducing eosinophilic inflammatory response in the treatment orprevention of asthma may be able to specifically address this feature ofthe disease. Also, elevated neutrophils, either in combination withelevated eosinophils or in their absence, is associated with severeasthma and chronic airway narrowing. Therefore, reducing theneutrophilic inflammatory response may be particularly useful foraddressing severe asthma.

In certain embodiments, the compositions reduce peribronchiolarinfiltration in allergic asthma, or are for use in reducingperibronchiolar infiltration in the treatment of allergic asthma. Incertain embodiments, the compositions reduce peribronchiolar and/orperivascular infiltration in neutrophilic asthma, or are for use inreducing peribronchiolar and/or perivascular infiltration in thetreatment of allergic neutrophilic asthma.

In certain embodiments, treatment with compositions of the inventionprovides a reduction or prevents an elevation in IL-1β, IFNγ, RANTES,MIP-1α or IL-8 levels.

In certain embodiments, the compositions of the invention are for use ina method of treating asthma that results in a reduction of theeosinophilic and/or neutrophilic inflammatory response. In certainembodiments, the patient to be treated has, or has previously beenidentified as having, elevated neutrophil or eosinophil levels, forexample as identified through blood sampling or sputum analysis.

The compositions of the invention may be useful for preventing thedevelopment of asthma in a new-born when administered to the new-born,or to a pregnant woman. The compositions may be useful for preventingthe development of asthma in children. The compositions of the inventionmay be useful for treating or preventing adult-onset asthma. Thecompositions of the invention may be useful for managing or alleviatingasthma. The compositions of the invention may be particularly useful forreducing symptoms associated with asthma that is aggravated byallergens, such as house dust mites.

Treatment or prevention of asthma may refer to, for example, analleviation of the severity of symptoms or a reduction in the frequencyof exacerbations or the range of triggers that are a problem for thepatient.

In certain embodiments, treatment with compositions of the inventionprovides a reduction in concentrations of phenylalanine and/orhistidine, for example in the intestines or in the plasma. The examplesdemonstrate that the bacterial strains of the compositions of theinvention tested positive for fermentation of amino acids, includingphenylalanine and histidine, and increased plasma concentrations ofphenylalanine and histidine have been reported to be associated withadverse effects in asthma. In certain embodiments, the compositions ofthe invention are for use in reducing plasma concentrations ofphenylalanine and/or histidine in the treatment of asthma, and inparticular in the treatment of histamine production or airwayhyper-responsiveness associated with asthma.

In certain embodiments, treatment with compositions of the inventionprovides a reduction in concentrations of galactose and/or fructose, forexample in the intestines. The examples demonstrate that the bacterialstrains of the compositions of the invention ferment carbohydratesubstrates including galactose and fructose, and galactoseα-1,3-galactose derived from meat sources is a known allergen andcausative agent of anaphylaxis, and intake levels of dietary fructoseare correlated with increased asthma severity. In certain embodiments,the compositions of the invention are for use in reducing concentrationsof galactose and/or fructose in the treatment of asthma, and inparticular in the treatment of severe asthma.

Arthritis

In preferred embodiments, the compositions of the invention are for usein treating or preventing rheumatoid arthritis (RA). The examplesdemonstrate that the compositions of the invention achieve a reductionin the clinical signs of RA in a mouse model, reduce cartilage and bonedamage, and reduce the IL-17 inflammatory response, and so they may beuseful in the treatment or prevention of RA. RA is a systemicinflammatory disorder that primarily affects joints. RA is associatedwith an inflammatory response that results in swelling of joints,synovial hyperplasia, and destruction of cartilage and bone. IL-17 andTh17 cells may have a key role in RA, for example because IL-17 inhibitsmatrix production in chondrocytes and osteoblasts and activates theproduction and function of matrix metalloproteinases and because RAdisease activity is correlated to IL-17 levels and Th-17 cell numbers[26,27], so the compositions of the invention may be particularlyeffective for preventing or treating RA.

In certain embodiments, the compositions of the invention are for use inlowering IL-17 levels or preventing elevation of IL-17 levels in thetreatment or prevention of RA. In certain embodiments, treatment withcompositions of the invention provides a reduction or prevents anelevation in IL-17 levels, in particular IL-17A levels. In certainembodiments, treatment with compositions of the invention provides areduction or prevents an elevation in IFN-γ or IL-6 levels.

In certain embodiments, treatment with the compositions of the inventionresults in a reduction in the swelling of joints. In certainembodiments, the compositions of the invention are for use in patientswith swollen joints or patients identified as at risk of having swollenjoints. In certain embodiments, the compositions of the invention arefor use in a method of reducing joint swelling in RA.

In certain embodiments, treatment with the compositions of the inventionresults in a reduction in cartilage damage or bone damage. In certainembodiments, the compositions of the invention are for use in reducingor preventing cartilage or bone damage in the treatment of RA. Incertain embodiments, the compositions are for use in treating patientwith severe RA that are at risk of cartilage or bone damage.

Increased IL-17 levels and Th17 cell numbers are associated withcartilage and bone destruction in RA [26,27]. IL-17 is known to activatematrix destruction in cartilage and bone tissue and IL-17 has aninhibitory effect on matrix production in chondrocytes and osteoblasts.Therefore, in certain embodiments, the compositions of the invention arefor use in preventing bone erosion or cartilage damage in the treatmentof RA. In certain embodiments, the compositions are for use in treatingpatients that exhibit bone erosion or cartilage damage or patientsidentified as at risk of bone erosion or cartilage damage.

TNF-α is also associated with RA, but TNF-α is not involved in thepathogenesis of the later stages of the disease. In contrast, IL-17 hasa role throughout all stages of chronic disease [28]. Therefore, incertain embodiments the compositions of the invention are for use intreating chronic RA or late-stage RA, such as disease that includesjoint destruction and loss of cartilage. In certain embodiments, thecompositions of the invention are for treating patients that havepreviously received anti-TNF-α therapy. In certain embodiments, thepatients to be treated do not respond or no longer respond to anti-TNF-αtherapy.

The compositions of the invention may be useful for modulating apatient's immune system, so in certain embodiments the compositions ofthe invention are for use in preventing RA in a patient that has beenidentified as at risk of RA, or that has been diagnosed with early-stageRA. The compositions of the invention may be useful for preventing thedevelopment of RA.

The compositions of the invention may be useful for managing oralleviating RA. The compositions of the invention may be particularlyuseful for reducing symptoms associated with joint swelling or bonedestruction. Treatment or prevention of RA may refer to, for example, analleviation of the severity of symptoms or a reduction in the frequencyof exacerbations or the range of triggers that are a problem for thepatient.

Multiple Sclerosis

In preferred embodiments, the compositions of the invention are for usein treating or preventing multiple sclerosis. The examples demonstratethat the compositions of the invention achieve a reduction in thedisease incidence and disease severity in a mouse model of multiplesclerosis (the EAE model), and so they may be useful in the treatment orprevention of multiple sclerosis. Multiple sclerosis is an inflammatorydisorder associated with damage to the myelin sheaths of neurons,particularly in the brain and spinal column. Multiple sclerosis is achronic disease, which is progressively incapacitating and which evolvesin episodes. IL-17 and Th17 cells may have a key role in multiplesclerosis, for example because IL-17 levels may correlate with multiplesclerosis lesions, IL-17 can disrupt blood brain barrier endothelialcell tight junctions, and Th17 cells can migrate into the centralnervous system and cause neuronal loss [29,30]. Therefore, thecompositions of the invention may be particularly effective forpreventing or treating multiple sclerosis.

In certain embodiments, treatment with the compositions of the inventionresults in a reduction in disease incidence or disease severity. Incertain embodiments, the compositions of the invention are for use inreducing disease incidence or disease severity. In certain embodiments,treatment with the compositions of the invention prevents a decline inmotor function or results in improved motor function. In certainembodiments, the compositions of the invention are for use in preventinga decline in motor function or for use in improving motor function. Incertain embodiments, treatment with the compositions of the inventionprevents the development of paralysis. In certain embodiments, thecompositions of the invention are for use in preventing paralysis in thetreatment of multiple sclerosis.

The compositions of the invention may be useful for modulating apatient's immune system, so in certain embodiments the compositions ofthe invention are for use in preventing multiple sclerosis in a patientthat has been identified as at risk of multiple sclerosis, or that hasbeen diagnosed with early-stage multiple sclerosis or“relapsing-remitting” multiple sclerosis. The compositions of theinvention may be useful for preventing the development of sclerosis.Indeed, the examples show that administration of compositions of theinvention prevented the development of disease in many mice.

The compositions of the invention may be useful for managing oralleviating multiple sclerosis. The compositions of the invention may beparticularly useful for reducing symptoms associated with multiplesclerosis. Treatment or prevention of multiple sclerosis may refer to,for example, an alleviation of the severity of symptoms or a reductionin the frequency of exacerbations or the range of triggers that are aproblem for the patient.

Uveitis

In preferred embodiments, the compositions of the invention are for usein treating or preventing uveitis. The compositions of the invention mayachieve a reduction in disease incidence and disease severity in ananimal model of uveitis and so they may be useful in the treatment orprevention of uveitis. Uveitis is inflammation of the uvea and canresult in retinal tissue destruction. It can present in differentanatomical forms (anterior, intermediate, posterior or diffuse) andresult from different, but related, causes, including systemicautoimmune disorders. IL-17 and the Th17 pathway are centrally involvedin uveitis, so the compositions of the invention may be particularlyeffective for preventing or treating uveitis. References [31-38]describe elevated serum levels of interleukin-17A in uveitis patients,specific association of IL17A genetic variants with panuveitis, the roleof Th17-associated cytokines in the pathogenesis of experimentalautoimmune uveitis, the imbalance between Th17 Cells and regulatory TCells during monophasic experimental autoimmune uveitis, theup-regulation of IL-17A in patients with uveitis and activeAdamantiades-Behcet and Vogt-Koyanagi-Harada (VKH) diseases, thetreatment of non-infectious uveitis with secukinumab (anti-IL-17Aantibody), and Th17 in uveitic eyes.

In certain embodiments, the uveitis is posterior uveitis. Posterioruveitis presents primarily with inflammation of the retina and choroidand the compositions of the invention may be effective for reducingretinal inflammation and damage.

In certain embodiments, treatment with the compositions of the inventionresults in a reduction in retinal damage. In certain embodiments, thecompositions of the invention are for use in reducing or preventingretinal damage in the treatment of uveitis. In certain embodiments, thecompositions are for use in treating patients with severe uveitis thatare at risk of retinal damage. In certain embodiments, treatment withthe compositions of the invention results in a reduction in optic discinflammation. In certain embodiments, the compositions of the inventionare for use in reducing or preventing optic disc inflammation. Incertain embodiments, treatment with the compositions of the inventionresults in a reduction in retinal tissue infiltration by inflammatorycells. In certain embodiments, the compositions of the invention are foruse in reducing retinal tissue infiltration by inflammatory cells. Incertain embodiments, treatment with the compositions of the inventionresults in vision being maintained or improved. In certain embodiments,the compositions of the invention are for use in maintaining orimproving vision.

In certain embodiments, the compositions are for use in treating orpreventing uveitis associated with a non-infectious or autoimmunedisease, such as Behcet disease, Crohn's disease, Fuchs heterochromiciridocyclitis, granulomatosis with polyangiitis, HLA-B27 relateduveitis, juvenile idiopathic arthritis, sarcoidosis, spondyloarthritis,sympathetic ophthalmia, tubulointerstitial nephritis and uveitissyndrome or Vogt-Koyanagi-Harada syndrome. IL-17A has been shown to beinvolved in, for example, Behcet and Vogt-Koyanagi-Harada diseases.

Treatment or prevention of uveitis may refer to, for example, analleviation of the severity of symptoms or a prevention of relapse.

Treating Cancer

In preferred embodiments, the compositions of the invention are for usein treating or preventing cancer. The examples demonstrate thatadministration of the compositions of the invention can lead to areduction in tumour growth in a number of tumour models.

In certain embodiments, treatment with the compositions of the inventionresults in a reduction in tumour size or a reduction in tumour growth.In certain embodiments, the compositions of the invention are for use inreducing tumour size or reducing tumour growth. The examples demonstratethat the compositions of the invention may be effective for reducingtumour size or growth. In certain embodiments, the compositions of theinvention are for use in patients with solid tumours. In certainembodiments, the compositions of the invention are for use in reducingor preventing angiogenesis in the treatment of cancer. IL-17 and Th17cells have central roles in angiogenesis. In certain embodiments, thecompositions of the invention are for use in preventing metastasis.

In certain embodiments, the compositions of the invention are for use intreating or preventing breast cancer. The examples demonstrate that thecompositions of the invention may be effective for treating breastcancer. In certain embodiments, the compositions of the invention arefor use in reducing tumour size, reducing tumour growth, or reducingangiogenesis in the treatment of breast cancer. In preferred embodimentsthe cancer is mammary carcinoma. In preferred embodiments the cancer isstage IV breast cancer.

In certain embodiments, the compositions of the invention are for use intreating or preventing lung cancer. The examples demonstrate that thecompositions of the invention may be effective for treating lung cancer.In certain embodiments, the compositions of the invention are for use inreducing tumour size, reducing tumour growth, or reducing angiogenesisin the treatment of lung cancer. In preferred embodiments the cancer islung carcinoma.

In certain embodiments, the compositions of the invention are for use intreating or preventing liver cancer. The examples demonstrate that thecompositions of the invention may be effective for treating livercancer. In certain embodiments, the compositions of the invention arefor use in reducing tumour size, reducing tumour growth, or reducingangiogenesis in the treatment of liver cancer. In preferred embodimentsthe cancer is hepatoma (hepatocellular carcinoma).

In certain embodiments, the compositions of the invention are for use intreating or preventing carcinoma. The examples demonstrate that thecompositions of the invention may be effective for treating numeroustypes of carcinoma. In certain embodiments, the compositions of theinvention are for use in treating or preventing non-immunogenic cancer.The examples demonstrate that the compositions of the invention may beeffective for treating non-immunogenic cancers.

IL-17 and the Th17 pathway have central roles in cancer development andprogression and, although the roles of IL-17 and Th17 cells in cancerare not fully understood, numerous pro-tumour effects of IL-17 and Th17cells are known. For example, Th17 cells and IL-17 can promoteangiogenesis, increase proliferation and survival of tumor cells andactivate tumour-promoting transcription factors [39-41]. Therefore, thecompositions of the invention may be useful for treating or preventingcancer. Furthermore, the examples demonstrate that the compositions ofthe invention are effective for reducing tumour volume in breast, lungand liver cancer, and IL-17 and Th17 cells have important roles in thesespecific types of cancer [42-44].

The therapeutic effects of the compositions of the invention on cancermay be mediated by a pro-inflammatory mechanism. Inflammation can have acancer-suppressive effect [45] and pro-inflammatory cytokines such asTNFα are being investigated as cancer therapies [46]. The compositionsof the invention may be useful for treating cancer via a similarmechanism. For example, the compositions of the invention may elicit anIFNγ-type response. IFNγ is a potent macrophage-activating factor thatcan stimulate tumirocidal activity [47], and CXCL9 and CXCL10, forexample, also have anti-cancer effects [48-50]. Therefore, in certainembodiments, the compositions of the invention are for use in promotinginflammation in the treatment of cancer. In preferred embodiments, thecompositions of the invention are for use in promoting Th1 inflammationin the treatment of cancer. Th1 cells produce IFNγ and have potentanti-cancer effects [45]. In certain embodiments, the compositions ofthe invention are for use in treating an early-stage cancer, such as acancer that has not metastasized, or a stage 0 or stage 1 cancer.Promoting inflammation may be more effective against early-stage cancers[45]. In certain embodiments, the compositions of the invention are foruse in promoting inflammation to enhance the effect of a secondanti-cancer agent.

In further embodiments, the compositions of the invention are for use intreating or preventing acute lymphoblastic leukemia (ALL), acute myeloidleukemia, adrenocortical carcinoma, basal-cell carcinoma, bile ductcancer, bladder cancer, bone tumor, osteosarcoma/malignant fibroushistiocytoma, brainstem glioma, brain tumor, cerebellar astrocytoma,cerebral astrocytoma/malignant glioma, ependymoma, medulloblastoma,supratentorial primitive neuroectodermal tumors, breast cancer,bronchial adenomas/carcinoids, Burkitt's lymphoma, carcinoid tumor,cervical cancer, chronic lymphocytic leukemia, chronic myelogenousleukemia, chronic myeloproliferative disorders, colon cancer, cutaneousT-cell lymphoma, endometrial cancer, ependymoma, esophageal cancer,Ewing's sarcoma, intraocular melanoma, retinoblastoma, gallbladdercancer, gastric cancer, gastrointestinal carcinoid tumor,gastrointestinal stromal tumor (GIST), germ cell tumor, glioma,childhood visual pathway and hypothalamic, Hodgkin lymphoma, melanoma,islet cell carcinoma, Kaposi sarcoma, renal cell cancer, laryngealcancer, leukaemias, lymphomas, mesothelioma, neuroblastoma, non-Hodgkinlymphoma, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreaticcancer, parathyroid cancer, pharyngeal cancer, pituitary adenoma, plasmacell neoplasia, prostate cancer, renal cell carcinoma, retinoblastoma,sarcoma, testicular cancer, thyroid cancer, or uterine cancer.

The compositions of the invention may be particularly effective whenused in combination with further therapeutic agents. Theimmune-modulatory effects of the compositions of the invention may beeffective when combined with more direct anti-cancer agents. Therefore,in certain embodiments, the invention provides a composition comprisingthe bacterium deposited under accession number NCIMB 42380 or a biotypethereof and an anticancer agent. In preferred embodiments the anticanceragent is an immune checkpoint inhibitor, a targeted antibodyimmunotherapy, a CAR-T cell therapy, an oncolytic virus, or a cytostaticdrug. In preferred embodiments, the composition comprises an anti-canceragent selected from the group consisting of: Yervoy (ipilimumab, BMS);Keytruda (pembrolizumab, Merck); Opdivo (nivolumab, BMS); MEDI4736(AZ/MedImmune); MPDL3280A (Roche/Genentech); Tremelimumab(AZ/MedImmune); CT-011 (pidilizumab, CureTech); BMS-986015 (lirilumab,BMS); MEDI0680 (AZ/MedImmune); MSB-0010718C (Merck); PF-05082566(Pfizer); MEDI6469 (AZ/MedImmune); BMS-986016 (BMS); BMS-663513(urelumab, BMS); IMP321 (Prima Biomed); LAG525 (Novartis); ARGX-110(arGEN-X); PF-05082466 (Pfizer); CDX-1127 (varlilumab; CellDexTherapeutics); TRX-518 (GITR Inc.); MK-4166 (Merck); JTX-2011 (JounceTherapeutics); ARGX-115 (arGEN-X); NLG-9189 (indoximod, NewLinkGenetics); INCB024360 (Incyte); IPH2201 (Innate Immotherapeutics/AZ);NLG-919 (NewLink Genetics); anti-VISTA (JnJ); Epacadostat (INCB24360,Incyte); F001287 (Flexus/BMS); CP 870893 (University of Pennsylvania);MGA271 (Macrogenix); Emactuzumab (Roche/Genentech); Galunisertib (EliLilly); Ulocuplumab (BMS); BKT140/BL8040 (Biokine Therapeutics);Bavituximab (Peregrine Pharmaceuticals); CC 90002 (Celgene); 852A(Pfizer); VTX-2337 (VentiRx Pharmaceuticals); IMO-2055 (Hybridon, IderaPharmaceuticals); LY2157299 (Eli Lilly); EW-7197 (Ewha Women'sUniversity, Korea); Vemurafenib (Plexxikon); Dabrafenib (Genentech/GSK);BMS-777607 (BMS); BLZ945 (Memorial Sloan-Kettering Cancer Centre);Unituxin (dinutuximab, United Therapeutics Corporation); Blincyto(blinatumomab, Amgen); Cyramza (ramucirumab, Eli Lilly); Gazyva(obinutuzumab, Roche/Biogen); Kadcyla (ado-trastuzumab emtansine,Roche/Genentech); Perjeta (pertuzumab, Roche/Genentech); Adcetris(brentuximab vedotin, Takeda/Millennium); Arzerra (ofatumumab, GSK);Vectibix (panitumumab, Amgen); Avastin (bevacizumab, Roche/Genentech);Erbitux (cetuximab, BMS/Merck); Bexxar (tositumomab-131, GSK); Zevalin(ibritumomab tiuxetan, Biogen); Campath (alemtuzumab, Bayer); Mylotarg(gemtuzumab ozogamicin, Pfizer); Herceptin (trastuzumab,Roche/Genentech); Rituxan (rituximab, Genentech/Biogen); volociximab(Abbvie); Enavatuzumab (Abbvie); ABT-414 (Abbvie); Elotuzumab(Abbvie/BMS); ALX-0141 (Ablynx); Ozaralizumab (Ablynx); Actimab-C(Actinium); Actimab-P (Actinium); Milatuzumab-dox (Actinium); Emab-SN-38(Actinium); Naptumonmab estafenatox (Active Biotech); AFM13 (Affimed);AFM11 (Affimed); AGS-16C3F (Agensys); AGS-16M8F (Agensys); AGS-22ME(Agensys); AGS-15ME (Agensys); GS-67E (Agensys); ALXN6000 (samalizumab,Alexion); ALT-836 (Altor Bioscience); ALT-801 (Altor Bioscience);ALT-803 (Altor Bioscience); AMG780 (Amgen); AMG228 (Amgen); AMG820(Amgen); AMG172 (Amgen); AMG595 (Amgen); AMG110 (Amgen); AMG232(adecatumumab, Amgen); AMG211 (Amgen/MedImmune); BAY20-10112(Amgen/Bayer); Rilotumumab (Amgen); Denosumab (Amgen); AMP-514 (Amgen);MEDI575 (AZ/MedImmune); MEDI3617 (AZ/MedImmune); MEDI6383(AZ/MedImmune); MEDI551 (AZ/MedImmune); Moxetumomab pasudotox(AZ/MedImmune); MEDI565 (AZ/MedImmune); MEDI0639 (AZ/MedImmune);MEDI0680 (AZ/MedImmune); MEDI562 (AZ/MedImmune); AV-380 (AVEO); AV203(AVEO); AV299 (AVEO); BAY79-4620 (Bayer); Anetumab ravtansine (Bayer);vantictumab (Bayer); BAY94-9343 (Bayer); Sibrotuzumab (BoehringerIngleheim); BI-836845 (Boehringer Ingleheim); B-701 (BioClin); BIIB015(Biogen); Obinutuzumab (Biogen/Genentech); BI-505 (Bioinvent); BI-1206(Bioinvent); TB-403 (Bioinvent); BT-062 (Biotest) BIL-010t (Biosceptre);MDX-1203 (BMS); MDX-1204 (BMS); Necitumumab (BMS); CAN-4 (Cantargia AB);CDX-011 (Celldex); CDX1401 (Celldex); CDX301 (Celldex); U3-1565 (DaiichiSankyo); patritumab (Daiichi Sankyo); tigatuzumab (Daiichi Sankyo);nimotuzumab (Daiichi Sankyo); DS-8895 (Daiichi Sankyo); DS-8873 (DaiichiSankyo); DS-5573 (Daiichi Sankyo); MORab-004 (Eisai); MORab-009 (Eisai);MORab-003 (Eisai); MORab-066 (Eisai); LY3012207 (Eli Lilly); LY2875358(Eli Lilly); LY2812176 (Eli Lilly); LY3012217 (Eli Lilly); LY2495655(Eli Lilly); LY3012212 (Eli Lilly); LY3012211 (Eli Lilly); LY3009806(Eli Lilly); cixutumumab (Eli Lilly); Flanvotumab (Eli Lilly); IM4C-TR1(Eli Lilly); Ramucirumab (Eli Lilly); Tabalumab (Eli Lilly); Zanolimumab(Emergent Biosolution); FG-3019 (FibroGen); FPA008 (Five PrimeTherapeutics); FP-1039 (Five Prime Therapeutics); FPA144 (Five PrimeTherapeutics); catumaxomab (Fresenius Biotech); IMAB362 (Ganymed);IMAB027 (Ganymed); HuMax-CD74 (Genmab); HuMax-TFADC (Genmab); GS-5745(Gilead); GS-6624 (Gilead); OMP-21M18 (demcizumab, GSK); mapatumumab(GSK); IMGN289 (ImmunoGen); IMGN901 (ImmunoGen); IMGN853 (ImmunoGen);IMGN529 (ImmunoGen); IMMU-130 (Immunomedics); milatuzumab-dox(Immunomedics); IMMU-115 (Immunomedics); IMMU-132 (Immunomedics);IMMU-106 (Immunomedics); IMMU-102 (Immunomedics); Epratuzumab(Immunomedics); Clivatuzumab (Immunomedics); IPH41 (InnateImmunotherapeutics); Daratumumab (Janssen/Genmab); CNTO-95 (Intetumumab,Janssen); CNTO-328 (siltuximab, Janssen); KB004 (KaloBios);mogamulizumab (Kyowa Hakko Kirrin); KW-2871 (ecromeximab, Life Science);Sonepcizumab (Lpath); Margetuximab (Macrogenics); Enoblituzumab(Macrogenics); MGD006 (Macrogenics); MGF007 (Macrogenics); MK-0646(dalotuzumab, Merck); MK-3475 (Merck); Sym004 (Symphogen/Merck Serono);DI17E6 (Merck Serono); MOR208 (Morphosys); MOR202 (Morphosys); Xmab5574(Morphosys); BPC-1C (ensituximab, Precision Biologics); TAS266(Novartis); LFA102 (Novartis); BHQ880 (Novartis/Morphosys); QGE031(Novartis); HCD122 (lucatumumab, Novartis); LJM716 (Novartis); AT355(Novartis); OMP-21M18 (Demcizumab, OncoMed); OMP52M51 (Oncomed/GSK);OMP-59R5 (Oncomed/GSK); vantictumab (Oncomed/Bayer); CMC-544 (inotuzumabozogamicin, Pfizer); PF-03446962 (Pfizer); PF-04856884 (Pfizer);PSMA-ADC (Progenics); REGN1400 (Regeneron); REGN910 (nesvacumab,Regeneron/Sanofi); REGN421 (enoticumab, Regeneron/Sanofi); RG7221,RG7356, RG7155, RG7444, RG7116, RG7458, RG7598, RG7599, RG7600, RG7636,RG7450, RG7593, RG7596, DCDS3410A, RG7414 (parsatuzumab), RG7160(imgatuzumab), RG7159 (obintuzumab), RG7686, RG3638 (onartuzumab),RG7597 (Roche/Genentech); SAR307746 (Sanofi); SAR566658 (Sanofi);SAR650984 (Sanofi); SAR153192 (Sanofi); SAR3419 (Sanofi); SAR256212(Sanofi), SGN-LIV1A (lintuzumab, Seattle Genetics); SGN-CD33A (SeattleGenetics); SGN-75 (vorsetuzumab mafodotin, Seattle Genetics); SGN-19A(Seattle Genetics) SGN-CD70A (Seattle Genetics); SEA-CD40 (SeattleGenetics); ibritumomab tiuxetan (Spectrum); MLN0264 (Takeda); ganitumab(Takeda/Amgen); CEP-37250 (Teva); TB-403 (Thrombogenic); VB4-845(Viventia); Xmab2512 (Xencor); Xmab5574 (Xencor); nimotuzumab (YMBiosciences); Carlumab (Janssen); NY-ESO TCR (Adaptimmune); MAGE-A-10TCR (Adaptimmune); CTL019 (Novartis); JCAR015 (Juno Therapeutics);KTE-C19 CAR (Kite Pharma); UCART19 (Cellectis); BPX-401 (BellicumPharmaceuticals); BPX-601 (Bellicum Pharmaceuticals); ATTCK20 (UnumTherapeutics); CAR-NKG2D (Celyad); Onyx-015 (Onyx Pharmaceuticals); H101(Shanghai Sunwaybio); DNX-2401 (DNAtrix); VCN-01 (VCN Biosciences);Colo-Adl (PsiOxus Therapeutics); ProstAtak (Advantagene); Oncos-102(Oncos Therapeutics); CG0070 (Cold Genesys); Pexa-vac (JX-594, JennerexBiotherapeutics); GL-ONC1 (Genelux); T-VEC (Amgen); G207 (Medigene);HF10 (Takara Bio); SEPREHVIR (HSV1716, Virttu Biologics); OrienX010(OrienGene Biotechnology); Reolysin (Oncolytics Biotech); SVV-001(Neotropix); Cacatak (CVA21, Viralytics); Alimta (Eli Lilly), cisplatin,oxaliplatin, irinotecan, folinic acid, methotrexate, cyclophosphamide,5-fluorouracil, Zykadia (Novartis), Tafinlar (GSK), Xalkori (Pfizer),Iressa (AZ), Gilotrif (Boehringer Ingelheim), Tarceva (Astellas Pharma),Halaven (Eisai Pharma), Veliparib (Abbvie), AZD9291 (AZ), Alectinib(Chugai), LDK378 (Novartis), Genetespib (Synta Pharma),Tergenpumatucel-L (NewLink Genetics), GV1001 (Kael-GemVax), Tivantinib(ArQule); Cytoxan (BMS); Oncovin (Eli Lilly); Adriamycin (Pfizer);Gemzar (Eli Lilly); Xeloda (Roche); Ixempra (BMS); Abraxane (Celgene);Trelstar (Debiopharm); Taxotere (Sanofi); Nexavar (Bayer); IMMU-132(Immunomedics); E7449 (Eisai); Thermodox (Celsion); Cometriq (Exellxis);Lonsurf (Taiho Pharmaceuticals); Camptosar (Pfizer); UFT (TaihoPharmaceuticals); and TS-1 (Taiho Pharmaceuticals).

Modes of Administration

Preferably, the compositions of the invention are to be administered tothe gastrointestinal tract in order to enable delivery to and/or partialor total colonisation of the intestine with the bacterial strain of theinvention. Generally, the compositions of the invention are administeredorally, but they may be administered rectally, intranasally, or viabuccal or sublingual routes.

In certain embodiments, the compositions of the invention may beadministered as a foam, as a spray or a gel.

In certain embodiments, the compositions of the invention may beadministered as a suppository, such as a rectal suppository, for examplein the form of a theobroma oil (cocoa butter), synthetic hard fat (e.g.suppocire, witepsol), glycero-gelatin, polyethylene glycol, or soapglycerin composition.

In certain embodiments, the composition of the invention is administeredto the gastrointestinal tract via a tube, such as a nasogastric tube,orogastric tube, gastric tube, jejunostomy tube (J tube), percutaneousendoscopic gastrostomy (PEG), or a port, such as a chest wall port thatprovides access to the stomach, jejunum and other suitable access ports.

The compositions of the invention may be administered once, or they maybe administered sequentially as part of a treatment regimen. In certainembodiments, the compositions of the invention are to be administereddaily.

In certain embodiments of the invention, treatment according to theinvention is accompanied by assessment of the patient's gut microbiota.Treatment may be repeated if delivery of and/or partial or totalcolonisation with the strain of the invention is not achieved such thatefficacy is not observed, or treatment may be ceased if delivery and/orpartial or total colonisation is successful and efficacy is observed.

In certain embodiments, the composition of the invention may beadministered to a pregnant animal, for example a mammal such as a humanin order to prevent an inflammatory or autoimmune disease developing inher child in utero and/or after it is born.

The compositions of the invention may be administered to a patient thathas been diagnosed with a disease or condition mediated by IL-17 or theTh17 pathway, or that has been identified as being at risk of a diseaseor condition mediated by IL-17 or the Th17 pathway. The compositions mayalso be administered as a prophylactic measure to prevent thedevelopment of diseases or conditions mediated by IL-17 or the Th17pathway in a healthy patient.

The compositions of the invention may be administered to a patient thathas been identified as having an abnormal gut microbiota. For example,the patient may have reduced or absent colonisation by the bacteriumdeposited under accession number NCIMB 42380.

The compositions of the invention may be administered as a food product,such as a nutritional supplement.

Generally, the compositions of the invention are for the treatment ofhumans, although they may be used to treat animals including monogastricmammals such as poultry, pigs, cats, dogs, horses or rabbits. Thecompositions of the invention may be useful for enhancing the growth andperformance of animals. If administered to animals, oral gavage may beused.

Compositions

Generally, the composition of the invention comprises bacteria. Inpreferred embodiments of the invention, the composition is formulated infreeze-dried form. For example, the composition of the invention maycomprise granules or gelatin capsules, for example hard gelatincapsules, comprising a bacterial strain of the invention.

Preferably, the composition of the invention comprises lyophilisedbacteria. Lyophilisation of bacteria is a well-established procedure andrelevant guidance is available in, for example, references [51-53].

Alternatively, the composition of the invention may comprise a live,active bacterial culture.

In preferred embodiments, the composition of the invention isencapsulated to enable delivery of the bacterial strain to theintestine. Encapsulation protects the composition from degradation untildelivery at the target location through, for example, rupturing withchemical or physical stimuli such as pressure, enzymatic activity, orphysical disintegration, which may be triggered by changes in pH. Anyappropriate encapsulation method may be used. Exemplary encapsulationtechniques include entrapment within a porous matrix, attachment oradsorption on solid carrier surfaces, self-aggregation by flocculationor with cross-linking agents, and mechanical containment behind amicroporous membrane or a microcapsule. Guidance on encapsulation thatmay be useful for preparing compositions of the invention is availablein, for example, references [54] and [55].

The composition may be administered orally and may be in the form of atablet, capsule or powder. Encapsulated products are preferred becausethe bacterium deposited under accession number NCIMB 42380 may be ananaerobe. Other ingredients (such as vitamin C, for example), may beincluded as oxygen scavengers and prebiotic substrates to improve thedelivery and/or partial or total colonisation and survival in vivo.Alternatively, the probiotic composition of the invention may beadministered orally as a food or nutritional product, such as milk orwhey based fermented dairy product, or as a pharmaceutical product.

The composition may be formulated as a probiotic.

A composition of the invention includes a therapeutically effectiveamount of a bacterial strain of the invention. A therapeuticallyeffective amount of a bacterial strain is sufficient to exert abeneficial effect upon a patient. A therapeutically effective amount ofa bacterial strain may be sufficient to result in delivery to and/orpartial or total colonisation of the patient's intestine.

A suitable daily dose of the bacteria, for example for an adult human,may be from about 1×10³ to about 1×10¹¹ colony forming units (CFU); forexample, from about 1×10⁷ to about 1×10¹⁰ CFU; in another example fromabout 1×10⁶ to about 1×10¹⁰ CFU.

In certain embodiments, the composition contains the bacterial strain inan amount of from about 1×10⁶ to about 1×10¹¹ CFU/g, respect to theweight of the composition; for example, from about 1×10⁸ to about 1×10¹⁰CFU/g. The dose may be, for example, 1 g, 3 g, 5 g, and 10 g.

Typically, a probiotic, such as the composition of the invention, isoptionally combined with at least one suitable prebiotic compound. Aprebiotic compound is usually a non-digestible carbohydrate such as anoligo- or polysaccharide, or a sugar alcohol, which is not degraded orabsorbed in the upper digestive tract. Known prebiotics includecommercial products such as inulin and transgalacto-oligosaccharides.

In certain embodiments, the probiotic composition of the presentinvention includes a prebiotic compound in an amount of from about 1 toabout 30% by weight, respect to the total weight composition, (e.g. from5 to 20% by weight). Carbohydrates may be selected from the groupconsisting of fructo-oligosaccharides (or FOS), short-chainfructo-oligosaccharides, inulin, isomalt-oligosaccharides, pectins,xylo-oligosaccharides (or XOS), chitosan-oligosaccharides (or COS),beta-glucans, arable gum modified and resistant starches, polydextrose,D-tagatose, acacia fibers, carob, oats, and citrus fibers. In oneaspect, the prebiotics are the short-chain fructo-oligosaccharides (forsimplicity shown herein below as FOSs-c.c); said FOSs-c.c. are notdigestible carbohydrates, generally obtained by the conversion of thebeet sugar and including a saccharose molecule to which three glucosemolecules are bonded.

The compositions of the invention may comprise pharmaceuticallyacceptable excipients or carriers. Examples of such suitable excipientsmay be found in the reference [56]. Acceptable carriers or diluents fortherapeutic use are well known in the pharmaceutical art and aredescribed, for example, in reference [57]. Examples of suitable carriersinclude lactose, starch, glucose, methyl cellulose, magnesium stearate,mannitol, sorbitol and the like. Examples of suitable diluents includeethanol, glycerol and water. The choice of pharmaceutical carrier,excipient or diluent can be selected with regard to the intended routeof administration and standard pharmaceutical practice. Thepharmaceutical compositions may comprise as, or in addition to, thecarrier, excipient or diluent any suitable binder(s), lubricant(s),suspending agent(s), coating agent(s), solubilising agent(s). Examplesof suitable binders include starch, gelatin, natural sugars such asglucose, anhydrous lactose, free-flow lactose, beta-lactose, cornsweeteners, natural and synthetic gums, such as acacia, tragacanth orsodium alginate, carboxymethyl cellulose and polyethylene glycol.Examples of suitable lubricants include sodium oleate, sodium stearate,magnesium stearate, sodium benzoate, sodium acetate, sodium chloride andthe like. Preservatives, stabilizers, dyes and even flavouring agentsmay be provided in the pharmaceutical composition. Examples ofpreservatives include sodium benzoate, sorbic acid and esters ofp-hydroxybenzoic acid. Antioxidants and suspending agents may be alsoused.

The compositions of the invention may be formulated as a food product.For example, a food product may provide nutritional benefit in additionto the therapeutic effect of the invention, such as in a nutritionalsupplement. Similarly, a food product may be formulated to enhance thetaste of the composition of the invention or to make the compositionmore attractive to consume by being more similar to a common food item,rather than to a pharmaceutical composition. In certain embodiments, thecomposition of the invention is formulated as a milk-based product. Theterm “milk-based product” means any liquid or semi-solid milk- orwhey-based product having a varying fat content. The milk-based productcan be, e.g., cow's milk, goat's milk, sheep's milk, skimmed milk, wholemilk, milk recombined from powdered milk and whey without anyprocessing, or a processed product, such as yoghurt, curdled milk, curd,sour milk, sour whole milk, butter milk and other sour milk products.Another important group includes milk beverages, such as whey beverages,fermented milks, condensed milks, infant or baby milks; flavoured milks,ice cream; milk-containing food such as sweets.

In certain embodiments, the compositions of the invention contain asingle bacterial strain or species and do not contain any otherbacterial strains or species. Such compositions may comprise only deminimis or biologically irrelevant amounts of other bacterial strains orspecies. Such compositions may be a culture that is substantially freefrom other species of organism.

The compositions for use in accordance with the invention may or may notrequire marketing approval.

In some cases, the lyophilised bacterial strain is reconstituted priorto administration. In some cases, the reconstitution is by use of adiluent described herein.

The compositions of the invention can comprise pharmaceuticallyacceptable excipients, diluents or carriers.

In certain embodiments, the invention provides a pharmaceuticalcomposition comprising: a bacterial strain of the invention; and apharmaceutically acceptable excipient, carrier or diluent; wherein thebacterial strain is in an amount sufficient to treat a disorder whenadministered to a subject in need thereof; and wherein the disorder isselected from the group consisting of asthma, allergic asthma,neutrophilic asthma, osteoarthritis, psoriatic arthritis, juvenileidiopathic arthritis, neuromyelitis optica (Devic's disease), ankylosingspondylitis, spondyloarthritis, systemic lupus erythematosus, celiacdisease, chronic obstructive pulmonary disease (COPD), cancer, breastcancer, colon cancer, lung cancer, ovarian cancer, uveitis, scleritis,vasculitis, Behcet's disease, atherosclerosis, atopic dermatitis,emphysema, periodontitis, allergic rhinitis, and allograft rejection.

In certain embodiments, the invention provides pharmaceuticalcomposition comprising: a bacterial strain of the invention; and apharmaceutically acceptable excipient, carrier or diluent; wherein thebacterial strain is in an amount sufficient to treat or prevent adisease or condition mediated by IL-17 or the Th17 pathway. In preferredembodiments, said disease or condition is selected from the groupconsisting of rheumatoid arthritis, multiple sclerosis, psoriasis,inflammatory bowel disease, Crohn's disease, ulcerative colitis, celiacdisease, asthma, allergic asthma, neutrophilic asthma, osteoarthritis,psoriatic arthritis, juvenile idiopathic arthritis, neuromyelitis optica(Devic's disease), ankylosing spondylitis, spondyloarthritis, systemiclupus erythematosus, chronic obstructive pulmonary disease (COPD),cancer, breast cancer, colon cancer, lung cancer, ovarian cancer,uveitis, scleritis, vasculitis, Behcet's disease, atherosclerosis,atopic dermatitis, emphysema, periodontitis, allergic rhinitis, andallograft rejection.

In certain embodiments, the invention provides the above pharmaceuticalcomposition, wherein the amount of the bacterial strain is from about1×10³ to about 1×10¹¹ colony forming units per gram with respect to aweight of the composition.

In certain embodiments, the invention provides the above pharmaceuticalcomposition, wherein the composition is administered at a dose of 1 g, 3g, 5 g or 10 g.

In certain embodiments, the invention provides the above pharmaceuticalcomposition, wherein the composition is administered by a methodselected from the group consisting of oral, rectal, subcutaneous, nasal,buccal, and sublingual.

In certain embodiments, the invention provides the above pharmaceuticalcomposition, comprising a carrier selected from the group consisting oflactose, starch, glucose, methyl cellulose, magnesium stearate, mannitoland sorbitol.

In certain embodiments, the invention provides the above pharmaceuticalcomposition, comprising a diluent selected from the group consisting ofethanol, glycerol and water.

In certain embodiments, the invention provides the above pharmaceuticalcomposition, comprising an excipient selected from the group consistingof starch, gelatin, glucose, anhydrous lactose, free-flow lactose,beta-lactose, corn sweetener, acacia, tragacanth, sodium alginate,carboxymethyl cellulose, polyethylene glycol, sodium oleate, sodiumstearate, magnesium stearate, sodium benzoate, sodium acetate and sodiumchloride.

In certain embodiments, the invention provides the above pharmaceuticalcomposition, further comprising at least one of a preservative, anantioxidant and a stabilizer.

In certain embodiments, the invention provides the above pharmaceuticalcomposition, comprising a preservative selected from the groupconsisting of sodium benzoate, sorbic acid and esters ofp-hydroxybenzoic acid.

In certain embodiments, the invention provides the above pharmaceuticalcomposition, wherein said bacterial strain is lyophilised.

In certain embodiments, the invention provides the above pharmaceuticalcomposition, wherein when the composition is stored in a sealedcontainer at about 4-C or about 25-C and the container is placed in anatmosphere having 50% relative humidity, at least 80% of the bacterialstrain as measured in colony forming units, remains after a period of atleast about: 1 month, 3 months, 6 months, 1 year, 1.5 years, 2 years,2.5 years or 3 years.

Culturing Methods

The bacterial strains for use in the present invention can be culturedusing standard microbiology techniques as detailed in, for example,references [58-60].

The solid or liquid medium used for culture may be YCFA agar or YCFAmedium. YCFA medium may include (per 100 ml, approximate values):Casitone (1.0 g), yeast extract (0.25 g), NaHCO₃ (0.4 g), cysteine (0.1g), K₂HPO₄ (0.045 g), KH₂PO₄ (0.045 g), NaCl (0.09 g), (NH₄)₂SO₄ (0.09g), MgSO₄.7H₂O (0.009 g), CaCl₂ (0.009 g), resazurin (0.1 mg), hemin (1mg), biotin (1 μg), cobalamin (1 μg), p-aminobenzoic acid (3 μg), folicacid (5 μg), and pyridoxamine (15 μg).

Bacterial Strains for Use in Vaccine Compositions

The inventors have identified that the bacterial strains of theinvention are useful for treating or preventing diseases or conditionsmediated by IL-17 or the Th17 pathway. This is likely to be a result ofthe effect that the bacterial strains of the invention have on the hostimmune system. Therefore, the compositions of the invention may also beuseful for preventing diseases or conditions mediated by IL-17 or theTh17 pathway, when administered as vaccine compositions. In certain suchembodiments, the bacterial strains of the invention may be killed,inactivated or attenuated. In certain such embodiments, the compositionsmay comprise a vaccine adjuvant. In certain embodiments, thecompositions are for administration via injection, such as viasubcutaneous injection.

General

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of chemistry, biochemistry, molecularbiology, immunology and pharmacology, within the skill of the art. Suchtechniques are explained fully in the literature. See, e.g., references[61] and [62-68], etc.

The term “comprising” encompasses “including” as well as “consisting”e.g. a composition “comprising” X may consist exclusively of X or mayinclude something additional e.g. X+Y.

The term “about” in relation to a numerical value x is optional andmeans, for example, x±10%.

The word “substantially” does not exclude “completely” e.g. acomposition which is “substantially free” from Y may be completely freefrom Y. Where necessary, the word “substantially” may be omitted fromthe definition of the invention.

References to a percentage sequence identity between two nucleotidesequences means that, when aligned, that percentage of nucleotides arethe same in comparing the two sequences. This alignment and the percenthomology or sequence identity can be determined using software programsknown in the art, for example those described in section 7.7.18 of ref.[69]. A preferred alignment is determined by the Smith-Waterman homologysearch algorithm using an affine gap search with a gap open penalty of12 and a gap extension penalty of 2, BLOSUM matrix of 62. TheSmith-Waterman homology search algorithm is disclosed in ref. [70].

Unless specifically stated, a process or method comprising numeroussteps may comprise additional steps at the beginning or end of themethod, or may comprise additional intervening steps. Also, steps may becombined, omitted or performed in an alternative order, if appropriate.

Various embodiments of the invention are described herein. It will beappreciated that the features specified in each embodiment may becombined with other specified features, to provide further embodiments.In particular, embodiments highlighted herein as being suitable, typicalor preferred may be combined with each other (except when they aremutually exclusive).

MODES FOR CARRYING OUT THE INVENTION Example 1—Efficacy of BacterialInocula in a Mouse Model of House Dust Mite-Induced Asthma

Summary

Mice were administered with compositions comprising bacterial strainsaccording to the invention and were subsequently challenged with housedust mite (HDM) extract to elicit an allergic inflammatory response. Theinflammatory response to HDM includes eosinophilic and neutrophiliccomponents, is mediated by IL-17 and the Th17 pathway, and is a modelfor asthma. The magnitude and characteristics of the inflammatoryresponse exhibited by mice treated with compositions of the inventionwere compared to control groups. The compositions of the invention werefound to alleviate the inflammatory response, and to reduce recruitmentof eosinophils and neutrophils, indicating that they may be useful fortreating IL-17-mediated conditions such as eosinophilia, neutrophiliaand asthma.

Strain

751: bacterium deposited under accession number NCIMB 42380

Study Design

Groups:

1. Negative control group. Treatment with vehicle control (per oral).

4. Treatment with therapeutic bacteria inoculum strain 751 (per oral).

7. Positive control group. Treatment with Dexamethasone (i.p.).

8. Untreated Control Group.

Number of mice per group=5

Day −14 to day 13: Daily administration of vehicle control per oral(Group 1).

Day −14 to day 13: Daily administration of therapeutic bacteria inoculumper oral (Group 2-6).

Day 0, 2, 4, 7, 9, 11 Administration of 15 ug HDM (house dust miteextract—Catalogue number: XPB70D3A25, Lot number: 231897, GreerLaboratories, Lenoir, N.C., USA) in a volume of 30 ul PBS per nasal(Group 1-8).

Day 0, 2, 4, 7, 9, 11 Administration of Dexamethasone (i.p., 3 mg/kg,Sigma-Aldrich, Catalogue number D1159) (Group 7).

Day 14 Sacrifice of all animals for analysis.

Total number of mice=40.

Endpoints and Analysis

On day 14 animals were sacrificed by lethal intraperitoneal injectionwith pentabarbitol (Streuli Pharma AG, Uznach, Cat: 1170139A)immediately followed by a bronchoalveolar lavage (BAL).

Cells were isolated from the BAL (bronchoalveolar lavage) fluid anddifferential cell counts performed (200 cell counts/samples).

Material and Methods

Mice. Female 7 week old BALB/c mice were purchased from Charles RiverLaboratories and randomly allocated to cages totally 5 mice per cage(Ventilated cages sourced from Indulab AG, Gams, Switzerland Cage type:“The Sealsafe™—IVC cage. Product number 1248L). Cages were labeled withstudy number, group number and experimental starting date. Mice weremonitored weekly and acclimatized to facility for 7 days prior toinitiation of study (Study Day −14). Animals were 8 weeks old on StudyDay −14. Potable water and food were available ad libitum. Cageenrichment was present. Daily care of the animals was performedaccording to local authorization license number 2283.1 (issued andapproved by: Service de la consommation et des affaires vétérinaires duCanton de Vaud). Potable water and food were available ad libitum andrefreshed once daily. Cage enrichment was present. Animal welfareregulations were observed as given by official authorities ofSwitzerland under ordinance 455.163 of the FVO (Federal VeterinaryOffice) on laboratory animal husbandry, production of geneticallymodified animals, and methods of animal experimentation.

Culturing of bacteria inoculum. Within a sterile workstation, acryo-vial of bacteria was thawed by warming in gloved hand and 0.7 ml ofcontents injected into a Hungate tube (Cat Number, 1020471,Glasgerätebau Ochs, Bovenden-Lenglern, Germany), containing 8 ml ofanaerobic YCFA. Two tubes per strain were usually prepared. The Hungatetubes were then incubated (static) at 37° C. for 16 h (strain 751).

Culturing of vehicle control. A Hungate tube containing 8 ml ofanaerobic YCFA was incubated (static) at 37° C. for 16 h.

Administration of bacteria inoculum or vehicle control. 400 ul ofcultured bacteria inoculum or vehicle control were administered per dayper oral gavage.

Intranasal sensitization. Mice were anesthetized by i.p. injection with9.75 mg xylasol and 48.75 mg ketasol per kg (Dr. E. Graeub A G, Bern,Switzerland) and administered with 15 ug of HDM (Catalogue number:XPB70D3A25, Lot number: 231897, Greer Laboratories, Lenoir, N.C., USA)in a volume of 30 ul PBS per nasal.

Preparation and administration of positive control compoundDexamethasone. Dexamethasone 21-phosphate disodium salt (Sigma-Aldrich,Catalogue number D1159, Lot No SLBD.1030V) was solved in H₂O andadministered to the animals in a dose of 3 mg/kg in a volume of 200 ulper oral at days indicated in study protocol above.

Terminal procedure. On day 14 animals were sacrificed by lethal i.p.injection with pentabarbitol (Streuli Pharma AG, Uznach, Cat: 1170139A)immediately followed by bronchoalveolar lavage (BAL) in 500 ul ofsaline.

Measurement of cellular infiltrates into BAL. Cells were isolated fromthe BAL fluid and differential cell counts were performed based uponstandard morphological and cytochemical criteria.

Graphs and statistical analysis. All graphs were generated with GraphpadPrism Version 6 and a one-way ANOVA was applied. Results from thestatistical analysis were provided with the individual data tables.Error bars represent Standard Error of the Mean (SEM).

Results and Analysis

The results of the experiments are shown in FIGS. 1-9.

No morbidity or mortality was noted in the mice treated with thebacteria or the vehicle. The two controls, vehicle treatment (negativecontrol) and the dexamethasone treatment (positive control) behaved asexpected, with impaired eosinophilia and neutrophilia noted followingdexamethasone treatment.

The most important results of this experiment are displayed in FIGS. 6and 7, which report on the total number and percentage of neutrophilsdetected in bronchiolar lavage following challenge with HDM. Strain 751reduced total neutrophils and the proportion of neutrophils in BALrelative to the vehicle-only control.

Example 2—Efficacy of Bacterial Inocula in a Mouse Model of SevereNeutrophilic Asthma

Summary

Mice were administered with compositions comprising bacterial strainsaccording to the invention and were subsequently sensitised withsubcutaneous administrations of house dust mite (HDM) extract andchallenged with an intranasal administration of HDM in order to modelthe inflammatory response of severe neutrophilic asthma. The magnitudeand characteristics of the inflammatory response exhibited by micetreated with compositions of the invention were compared to controlgroups. The compositions of the invention were found to alleviate theinflammatory response, and in particular to reduce recruitment ofneutrophils, in a manner comparable to the positive control comprisingadministrations of anti-IL-17 antibodies. The data therefore indicatethat the compositions of the invention may be useful for treating IL-17-and Th17-mediated conditions such as neutrophilia and asthma.

Strain

751: bacterium deposited under accession number NCIMB 42380

Study Design

Groups:

1. Negative control group. Treatment with vehicle control (per oral).

4. Treatment with therapeutic bacteria inoculum strain 751 (per oral).

7. Positive control group. Treatment anti-IL-17 (i.p.).

8. Untreated Control Group.

9: Healthy mice (baseline).

Number of mice per group (Group 1-8)=5

Day −14 to day 17: Daily administration of vehicle control per oral(Group 1).

Day −14 to day 17: Daily administration of therapeutic bacteria inoculumper oral (Group 2-6).

Day 0: Sensitization with HDM in CFA (s.c.) (Group 1-8).

Day 7: Sensitization with HDM in CFA (s.c.) (Group 1-8).

Day 13, 15, 17: Administration of anti IL-17 neutralizing antibody peri.p. (Group 7).

Day 14, 15, 16, 17: Challenge with HDM in 30 ul PBS per nasal (Group1-8).

Day 18: Sacrifice of all animals for analysis.

Endpoints and Analysis:

On day 14 animals were sacrificed by lethal intraperitoneal injectionwith pentabarbitol (Streuli Pharma AG, Uznach, Cat: 1170139A)immediately followed by a bronchoalveolar lavage (BAL). Cells wereisolated from the BAL fluid and differential cell counts performed (200cell counts/samples).

Material and Methods.

Mice. Female 7 week old C57BL/6 mice were purchased from Charles RiverLaboratories and randomly allocated to cages totally 5 mice per cage(Ventilated cages sourced from Indulab AG, Gams, Switzerland Cage type:“The Sealsafe™—IVC cage. Product number 1248L). Cages were labelled withstudy number, group number and experimental starting date. Mice weremonitored weekly and acclimatized to facility for 7 days prior toinitiation of study (Study Day −14). Animals were 8 weeks old on StudyDay −14. Potable water and food were available ad libitum. Cageenrichment was present. Daily care of the animals was performedaccording to local authorization license number 2283.1 (issued andapproved by: Service de la consommation et des affaires vétérinaires duCanton de Vaud). Potable water and food were available ad libitum andrefreshed once daily. Cage enrichment was present. Animal welfareregulations were observed as given by official authorities ofSwitzerland under ordinance 455.163 of the FVO (Federal VeterinaryOffice) on laboratory animal husbandry, production of geneticallymodified animals, and methods of animal experimentation.

Culturing of bacteria inoculum. Within a sterile workstation, acryo-vial of bacteria was thawed by warming in gloved hand and 0.7 ml ofcontents injected into a Hungate tube (Cat Number, 1020471,Glasgerätebau Ochs, Bovenden-Lenglern, Germany), containing 8 ml ofanaerobic YCFA. Two tubes per strain were usually prepared. The Hungatetubes were then incubated (static) at 37° C. for 16 h (strain 751).

Culturing of vehicle control. A Hungate tube containing 8 ml ofanaerobic YCFA was incubated (static) at 37° C. for 16 h.

Administration of bacteria inoculum or vehicle control. 400 ul ofcultured bacteria inoculum or vehicle control were administered per dayper oral gavage.

HDM sensitization. 50 μg of HDM (Catalogue number: XPB70D3A25, Lotnumber: 231897, Greer Laboratories, Lenoir, N.C., USA) in PBS wasemulsified in equal volume of complete Freund's adjuvant (CFA ChondrexInc. Washington, USA) and administered subcutaneously in a volume of 200μl, twice over two weeks on opposite flanks. A week after the secondimmunization, mice were anesthetized by i.p. injection with 9.75 mgxylasol and 48.75 mg ketasol per kg (Dr. E. Graeub A G, Bern,Switzerland) and then given intranasal challenges of 15 μg of HDM in avolume of 30 ul PBS on 4 consecutive days. Analysis was performed oneday after the final challenge.

Preparation and administration of positive control compound anti mouseIL-17 antibody. Anti-IL-17 neutralizing antibody was sourced from Bio XCell and was stored at 4° C. (Clone 17F3, Cat. Number BE0173, Bio XCell) and administered per i.p. at a dose of 12.5 mg/kg at daysindicated in study protocol above.

Terminal procedure. On day 18 animals were sacrificed by lethal i.p.injection with pentabarbitol (Streuli Pharma AG, Uznach, Cat: 1170139A)immediately followed by bronchoalveolar lavage (BAL) in 500 ul ofsaline.

Measurement of cellular infiltrates into BAL. Cells were isolated fromthe BAL fluid and differential cell counts were performed based uponstandard morphological and cytochemical criteria.

Graphs and statistical analysis. All graphs were generated with GraphpadPrism Version 6 and a one-way ANOVA was applied. Results from thestatistical analysis are provided with the individual data tables. Errorbars represent Standard Error of the Mean (SEM).

Results and Analysis

The results of the experiment are shown in FIGS. 10-18.

No morbidity or mortality was noted in the mice treated with thebacteria or the vehicle. As shown in FIGS. 15 and 16, strain 751 washighly efficacious in alleviating the magnitude of the neutrophilicinflammatory response. Indeed, the treatment with strain 751 showedcomparable results to treatment with anti-IL-17 antibodies. In addition,strain 751 reduced eosinophil numbers relative to the controls, as shownin FIGS. 11 and 12.

Example 3—Efficacy of Bacterial Inocula to Treat Arthritis in a Type IICollagen-Induced Arthritis Mouse Model

Materials and Methods

Strains

751: bacterium deposited under accession number NCIMB 42380

Bacterial Cultures

Bacterial cultures were grown up for administration in an anaerobicworkstation (Don Whitley Scientific).

Bacterial strain #751 was grown using glycerol stocks. The glycerolstocks were stored at −80° C. Three times per week, glycerol stocks werethawed at room temperature and streaked on YCFA plates. A new glycerolaliquot was used on each occasion. Bacteria were allowed to grow on agiven plate for up to 72 hours.

Solutions to be administered to the animals were prepared twice dailywith an eight hour interval for morning (AM) and afternoon (PM)treatments. A bacterial colony was picked from the streaked plate andtransferred into a tube containing YCFA media. Bacterial strain #751 wasallowed to grow for 16 hours before AM administrations. Bacteria weresub-cultured at 1% into YCFA media for PM administrations. OD valueswere recorded for each strain after morning and afternoon treatmentpreparations.

Type II Collagen-Induced Arthritis Mouse Model

Adult male DBA/1 mice were randomly allocated to experimental groups andallowed to acclimatise for two weeks. On Day 0, animals wereadministered by subcutaneous injection with 100 microliters of anemulsion containing 100 micrograms of type II collagen (CII) inincomplete's Freund's adjuvant supplemented with 4 mg/ml Mycobacteriumtuberculosis H37Ra. On Day 21, animals were administered by subcutaneousinjection with a booster emulsion containing 100 g of type II collagenin incomplete Freund's adjuvant.

Treatments were given according to the administration schedule below.From Day −14 until the end of the experiment on Day 45, animals wereweighed three times per week. From Day 21 until the end of theexperiment, animals were scored three times per week for clinical signsof arthritis to include swelling of the hind- and front paws,radio-carpal (wrist) joints and tibio-tarsal (ankle) joints.

On Day 45 mice were culled and terminal blood samples were taken forcytokine analysis.

On Day −14, Day 0 and Day 45, faecal samples were collected formicrobiological analysis, immediately snap-frozen and stored at −80° C.

The collagen-induced arthritis (CIA) mouse model is a well-establishedmouse model for rheumatoid arthritis [71]. Immunisation with CII causesa pathogenesis that includes several important pathological features ofrheumatoid arthritis, including synovial hyperplasia, mononuclear cellinfiltration and cartilage degradation. Significantly, the developmentof CIA is mediated by Th17 cells through secretion of IL-17A [72]. Theimmune response underlying the arthritis model is enhanced by the use ofFreund's adjuvant supplemented with Mycobacterium tuberculosis.

On Day 21, spleens were collected from three satellite animals in eachgroup. Cells were cultured for 72 hours in the presence or absence oftype II collagen. Cytokines, including TNF-α, IL-6, IFN-γ, IL-4, IL-10and IL-17, were quantified in the culture supernatants and in terminalserum by Luminex. Cell proliferation was quantified using a tritiatedthymidine incorporation method.

Treatment Groups and Dosages

All Groups were n=15 (n=12 for the main study group and n=3 forsatellite groups)

The vehicle used for the biotherapeutics was Yeastextract-Casitone-Fatty Acids (YCFA) medium.

Administration Disease Group Dose Route Regimen Induction 1 Vehicle 5ml/kg PO BID: Day 0: Day-14-End Collagen/CFA, once, SC 2 Biotherapeutic5 ml/kg PO BID: Day 21: #751 Day-14-End Collagen/IFA, once, SC PO: oralgavage, SC: subcutaneous injection, BID: twice a day, CFA: completeFreund’s adjuvant.Bodyweights

From Day −14 until the end of the experiment, animals were weighed threetimes per week. Data were graphed (Mean±SEM).

Non-Specific Clinical Observations

From Day −14 until the end of the experiment, animals were checked dailyfor non-specific clinical signs to include abnormal posture (hunched),abnormal coat condition (piloerection) and abnormal activity levels(reduced or increased activity).

Clinical Observations

From Day 21 until the end of the experiment on Day 45, animals werescored three times per week for clinical signs of arthritis to includeswelling of the hind- and front paws, radio-carpal (wrist) joints andtibio-tarsal (ankle) joints. Each limb was scored using the followingscale: (0) normal, (1) slight swelling, (2) mild swelling, (3) moderateswelling and (4) severe swelling. A clinical score was calculated byadding each limb score. The maximum possible clinical score for ananimal was (16). Animals with a score equal to (12) on two consecutiveoccasions and animals with a score greater than (12) on any one occasionwere culled. Data were graphed (Mean±SEM).

Cell Proliferation Analysis

On Day 21, three satellite animals per group were culled and spleenswere dissected out. Spleen cells were cultured for 72 hours in presenceor absence of type II Collagen. After 72 hours, cells were pulsedovernight in the presence of tritiated thymidine. Cell proliferation wasquantified by measuring thymidine incorporation. Data were graphed(Mean±SEM). Supernatants were taken and tested for the presence of keycytokines.

Cytokine Analysis

Terminal supernatants from the spleen cell cultures were tested in orderto quantitate TNF-α, IL-6, IFN-γ, IL-4, IL-10 and IL-17 by Luminex. Datawere graphed (Mean±SEM).

Microbiological Analysis

On Day −14, Day 0 and Day 45, faecal samples were collected from eachanimal, immediately snap-frozen, and stored at −80° C. Caeca (includingcontent) were immediately snap-frozen and stored at −80° C. A bacterialidentification test was performed daily by plating the bacteria.

Histopathology

At the end of the experiment, hind paws were stored in tissue fixative.Samples were transferred into decalcification solution. Tissue sampleswere processed, sectioned and stained with Haematoxylin & Eosin.Sections were scored by a qualified histopathologist, blind to theexperimental design, for signs of arthritis to include inflammation,articular cartilage damage and damage to the underlying metaphysealbone. A detailed scoring system was used (see below). Data were graphed(Mean±SEM). Raw and analysed data were provided as well asrepresentative pictures.

TABLE 1 Histopathology Scoring System Grade Description Inflammation 0Normal joint 1 Mild synovial hyperplasia with inflammation dominated byneutrophils. Low numbers of neutrophils and macrophages in joint space.2 Synovial hyperplasia with moderate to marked inflammation involvingboth neutrophils and macrophages. Neutrophils and macrophages in jointspace; may be some necrotic tissue debris. 3 Synovial hyperplasia withmarked inflammation involving both neutrophils and macrophages. Loss ofsynoviocyte lining. Inflammation may extend from synovium to surroundingtissue including muscle. Numerous neutrophils and macrophages in jointspace, together with significant necrotic tissue debris. Articularcartilage damage 0 Normal joint 1 Articular cartilage shows only milddegenerative change. Early pannus formation may be present peripherally.2 Articular cartilage shows moderate degenerative change and focal loss.Pannus formation is present focally. 3 Significant disruption and lossof articular cartilage with extensive pannus formation. Damage to theunderlying metaphyseal bone 0 Normal joint 1 No change to underlyingmetaphyseal bone. 2 May be focal necrosis or fibrosis of metaphysealbone. 3 Disruption or collapse of metaphyseal bone. Extensiveinflammation, necrosis or fibrosis extending to medullary space of themetaphysis.Results and AnalysisSurvival and Non-Specific Clinical Observations

Some animals were culled prior to the scheduled end of the study due tothe severity of the clinical signs of arthritis or due to the severityof the non-specific clinical observations.

Three animals were culled or found dead or culled during thepre-treatment period (Day −14 to Day 0): one animal in Group 1(vehicle-treated, animal arrived from supplier with broken leg and wasculled) and two animals in Group 2 (biotherapeutic #751-treated,possible lung dosing on first pre-treatment day, and post-dose clinicalsigns on second pre-treatment day).

Eight animals were culled due to the severity of the clinical signs ofarthritis: five animals in Group 1 (vehicle-treated) and three animalsin Group 2 (biotherapeutic #751-treated).

Four animals were culled due to the severity of the non-specificclinical signs including abnormal posture (hunched), abnormal coatcondition (piloerection), abnormal activity levels (reduced activity):three animals in Group 1 (vehicle-treated) and one animal in Group 2(biotherapeutic #751-treated).

Bodyweights

Bodyweight data recorded from Day −14 until Day 0 and expressed as apercentage of the initial (Day −14) bodyweights were analysed by two-wayANOVA followed by Dunnett's post-test for multiple comparisons with Day−14 then for multiple comparison with the vehicle-treated group. Thedata are presented in FIG. 19. Data from animals culled prior to thescheduled end of the experiment were excluded from the analyses.

When compared to Day −14, twice daily administrations by oral gavageinduced a significant bodyweight loss in the vehicle-treated group onDay −9 and Day −7.

The bodyweights measured between Day −14 and Day −1 in thebiotherapeutic-treated groups did not differ from the bodyweightsmeasured in the vehicle-treated group on any given day.

Bodyweight data recorded from Day 0 until Day 28 and expressed as apercentage of the initial (Day 0) bodyweights were analysed by two-wayANOVA followed by Dunnett's post-test for multiple comparisons with Day0 in the Vehicle group then for multiple comparison with thevehicle-treated group. The data are presented in FIG. 20. Data fromanimals culled prior to the scheduled end of the experiment and fromSatellite animals were excluded from the analyses. Day 28, Day 35 andDay 42 data were further analysed by one-way ANOVA followed by Dunnett'spost-test for multiple comparisons to the vehicle-treated group.

The onset of clinical signs of arthritis was associated with asignificant bodyweight loss on Day 26 and Day 28 (p<0.0001) whencompared to Day 0 in the vehicle-treated group.

There was no significant difference between experimental groups on Day35 or Day 42.

Clinical Observations

Clinical score data were analysed by two-way ANOVA followed by Dunnett'spost-test for multiple comparisons between days in the vehicle-treatedgroup then for multiple comparisons between experimental groups and thevehicle-treated group each day. The data are presented in FIG. 21. Datarecorded from animals culled prior to the end of the experiment wereexcluded from the analysis. When animals were culled due to the severityof the clinical signs of arthritis, the last recorded score was reportedfor the following days and used in the statistical analyses.

A significant increase of the clinical scores was observed in thevehicle-treated group from Day 28 until Day 45 (p<0.0001) when comparedto Day 21.

Biotherapeutic #751 induced a reduction of the clinical scores whencompared to the vehicle-treated group from Day 31 until Day 45, althoughthe difference was non-significant.

Cell Proliferation Analysis

To validate the assay, splenocytes were cultured in the presence ofsoluble anti-CD3 and anti-CD28 (anti-CD3/CD28) as positive controlstimuli to confirm the proliferative potential of the cells.

Strong proliferative responses to anti-CD3/CD28 were seen in allexperimental groups, showing cells were healthy, viable and able torespond to activation signals.

To test the proliferative response in presence of Collagen II (CII),splenocytes were cultured in the presence of CII at 50 μg/ml. Splenocyteproliferative response to CII were analysed by two-way ANOVA followed bySydak's post-test for multiple comparisons between unstimulated andCII-stimulated splenocytes and one-way ANOVA followed by Dunnett'spost-test for comparison of CII-stimulated response in differentexperimental groups with the vehicle-treated group. The data arepresented in FIG. 22.

CII induced a highly significant increase of ³H-thymidine incorporation(cpm) when compared to the unstimulated splenocytes in thevehicle-treated group (p<0.0001).

The groups treated with biotherapeutic #751 demonstrated significantlylower levels of CII-induced splenocyte proliferation than thevehicle-treated group.

Cytokine Levels in Tissue Culture Supernatants

Levels of each cytokine were measured in tissue culture supernatantsderived from anti-CD3/CD28 stimulated cultures by luminex analysis.These showed robust responses for all cytokines measured (mean levels invehicle group were as follows: IL-4=6,406 μg/ml; IL-6=306 μg/ml;IL-10=10,987 μg/ml; IL-17A=11,447 μg/ml; IFN-γ=15,581 μg/ml; TNF-α=76μg/ml).

The following sections summarise the data obtained from the CollagenII-stimulated cultures. Where applicable, statistical analyses of thedifferences between cytokine levels in supernatants of unstimulated andCII-stimulated splenocytes were conducted using two-way ANOVA followedby Sidak's post-test for multiple comparisons, while one-way ANOVAfollowed by Dunnett's post-test was used for comparison ofCII-stimulated response in biotherapeutic-treated groups with thevehicle-treated group. There was no significant difference in cytokinelevels between the groups in both cases. This is likely due to the smallsample size used (n=3).

In order to more accurately present the distribution of the data for thecytokines with substantial spread of the data, these are presented asscatter plots.

The group means of IL-4 in tissue culture supernatants after stimulationwith CII were <5 μg/ml. These are not considered biologicallysignificant and not included here. The group means of TNF-α in tissueculture supernatants after stimulation with collagen were below limit ofquantitation.

Supernatant Levels of IFN-γ (FIG. 23)

Along with IL-17, IFN-γ is the major cytokine driving disease in the CIAmodel. The scatter plot in FIG. 23 demonstrates IFN-γ levels after CIIstimulation, with group median being higher for the Vehicle-treatedgroup compared to the biotherapeutic. The outlier result from the samegroup 2 subject is responsible for the higher median in this group forIFN-γ and IL-10.

Supernatant Levels of IL-17A (FIG. 24)

Levels of IL-17A were 50 μg/ml in C-stimulated cultures for theVehicle-treated group. The levels of this cytokine appeared to be lowerin the biotherapeutic group compared to the Vehicle-treated.

Supernatant Levels of IL-10 (FIG. 25)

Levels of IL-10 in Vehicle-treated group were 13 μg/ml and 2.1 μg/ml forCII-stimulated, and media control cultures, respectively. Higher levelsof IL-10 (which is an anti-inflammatory cytokine) for thevehicle-treated group may be expected because inflammation andpro-inflammatory cytokine induction could be accompanied by ananti-inflammatory feedback mechanism.

Supernatant Levels of IL-6 (FIG. 26)

Inflammatory cytokines such as IL-6 and TNF-α are not typically producedat high levels in anti-CII cultures. However, their levels may bealtered as a result of immune modulation. Levels of IL-6 inCII-stimulated cultures were modest, reaching 10 μg/ml. Although higherthan in media control cultures, these differences were too small toprovide rationale for performing statistical analyses.

Microbiological Analysis

Bacterial growth was confirmed by measuring the optical density at 600nm using a spectrophotometer. Bacterial identity was confirmed bycomparing streaked plate pictures to reference pictures.

Following the improved bacterial preparation method, consistently highdoses of bacterial strain were administered from Day −2 and Day −3 asindicated by the high OD values measured.

Faecal samples were collected and snap-frozen on Day −14, Day 0 and attermination.

Histopathology

The histopathology results are shown in FIGS. 66-70. As expected forthis model, intra-individual and inter-individual variability wasobserved in terms of the presence/absence of arthritis or the severityof change present.

The nature of the pathology was as expected for this model, withextensive mixed chronic-active inflammation of the synovium and bursaextending to involve the peri-articular soft tissues (muscle, adiposetissue, dermal collagen). In the most severely affected joints there wasarticular cartilage degeneration and loss with intra-articular debrisand inflammation and disruption of the joint and bone structure byfibrosis and inflammation.

The incidence of histopathological changes was: vehicle—80% (16/20);Biotherapeutic #751-45% (9/20). Treatment with Biotherapeutic #751reduced the incidence of histopathological scores in mouse hind limbswhen compared to the vehicle-treated group (see FIGS. 66-69).Histopathology scores were analysed by one-way ANOVA for non-parametricdata (Kruskal-Wallis test) followed by Dunn's post-test for multiplecomparisons to the vehicle-treated group. Biotherapeutic #751 induced asignificant reduction of the joint inflammation scores observed inhistopathology when compared to the vehicle-treated group (p<0.01).Biotherapeutic #751 induced a significant reduction of the cartilagedamage scores observed in histopathology when compared to thevehicle-treated group (p<0.001). Biotherapeutic #751 induced asignificant reduction of the bone damage scores observed inhistopathology when compared to the vehicle-treated group (p<0.001).Biotherapeutic #751 induced a significant reduction of the totalhistopathology scores when compared to the vehicle-treated group(p<0.01).

Summary

Increased clinical scores were observed from Day 28 after the firstadministration of type II collagen, as expected in this model ofarthritis in DBA/1 mice. Biotherapeutic #751 was shown to be effectiveat treating arthritis in this model and Biotherapeutic #751 waseffective for reducing the severity of the clinical scores.Biotherapeutic #751 was also effective for reducing pathological diseasein the joints, as demonstrated in the histopathological analysis.

Proliferative recall responses to Collagen II were seen in splenocytecultures from all experimental groups. The collagen-specific responsewas significantly reduced following treatment with biotherapeutic #751(Group 2).

Most of the T cell cytokines tested showed detectable increases betweenCollagen II-stimulated and media controls in the Vehicle-treated group.These increases were not as obvious in the biotherapeutic-treated group.This broadly supports the proliferative recall responses to Collagen IIdescribed above.

There was evidence of suppression of the Th1/Th17 axis, which is thepathogenic response in this model and in human RA. Correlation ofreduced levels of cytokines with reduced proliferation is suggestive ofimmune modulation. There was no evidence that this modulation resultedeither from enhanced levels of Th2 associated IL-4 or with increases inthe immune modulating cytokine, IL-10.

Example 4—Further Analysis of the Effect of Bacterial Inocula in theMouse Model of House Dust Mite-Induced Asthma

The mice tested in Example 1 were subjected to further analyses tofurther characterise the effect of the compositions of the invention onthe allergic asthma inflammatory response.

Materials and Methods

Blood withdrawal and serum preparation on day 14. Blood samples ofanimals were collected via cardiac puncture. Serum was isolated from theblood sample by centrifugation for 5 min at 14000 g and stored at −20°C.

Organ removal on day 14. Collection of the left lung lobe in formalinfor follow-on histological analysis. Collection of the right lung lobes(all remaining lobes) and removal of serum for snap freezing andfollow-on analysis. Remaining BAL fluid was snap frozen for follow-onanalysis.

Measurement of Antibody Levels in Serum and BAL Fluid

Total IgE and house-dust-mite (HDM) specific IgG1 antibody productionwere measured in the BAL and serum by ELISA assay.

Isolation of Lung and Histological Analysis

Left lung lobes were fixed in formalin followed by embedment inparaffin, sectioning, and staining with hematoxylin and eosin and PAS.Subsequent histological scoring was performed blinded as followed: Fiverandom fields of view per sample were scored for inflammation(peribronchial infiltration and perivascular infiltration) and mucusproduction. Inflammatory infiltration was scored with the followinggrading system:

0—normal

1—mild inflammatory infiltrates

2—moderate inflammatory infiltrates

3—marked inflammatory infiltrates

4—severe inflammatory infiltrates

5—very severe inflammatory infiltrates

In each field of view, airways were measured in size and mucus cellnumbers were quantified/um.

Measurement of Inflammatory Mediators in Lung Tissue

Right lung lobes (all remaining lobes) isolated for quantification ofinflammatory mediators were snap frozen for subsequent measurement ofCCL11, IFN-gamma, IL-1 alpha, IL-1 beta, IL-4, IL-5, IL-9, IL-17A,CXCL1, CCL3, CXCL2 and CCL5 by commercially available multiplex assay(Merck-Millipore). Analysis was performed according to themanufacturer's instructions.

Results and Analysis

The results of the experiments are shown in FIGS. 28-46.

In support of the findings described in Example 1, analysis of thecellular infiltrates in the lung tissue of mice treated with strain 751showed a notable and statistically significant reduction in meaninflammation score (see FIGS. 32 and 34).

Antibody levels in the BAL fluid and serum were analysed (see FIGS.28-31). No clear effect of the bacterial treatment on serum antibodylevels was observed. This may reflect a failure in the experiment,because the spread of data and the error bars for each treatment arelarge, and the positive and negative controls do not appear to havebehaved as would be expected. Also, the baseline serum antibody levelscould have masked any changes.

Similarly, no clear effect of the bacterial treatment on cytokine levelsin lung tissue was observed (see FIGS. 36-46). Again, this may reflect afailure in the experiment, because the spread of data and the error barsfor each treatment are large, and the positive and negative controls donot appear to have behaved as would be expected. It is also possiblethat the mechanism of action involved influences earlier cytokineresponses that were no longer detectable on day 4 post the final HDMairway challenge. Some care should be taken when interpreting thecytokine data in the current study, due to the variability in the levelsdetected. This variability could in part be explained by the fact thatthe lung tissue was separated for the different analyses, and thus onelung lobe might not have been fully representative or comparable to thesame lobe in other mice due to patchy distribution of the inflammation.

Example 5—Further Analysis of the Effect of Bacterial Inocula in theMouse Model of Severe Neutrophilic Asthma

The mice tested in Example 2 were subjected to further analyses tofurther characterise the effect of the compositions of the invention onthe neutrophilic response associated with severe asthma.

Materials and Methods

Organ removal on day 18. Collection of the left lung lobe in formalinfor follow-on histological analysis. Collection of the right lung lobes(all remaining lobes) and removal of serum for snap freezing andfollow-on analysis. Remaining BAL fluid was snap frozen for follow-onanalysis.

Measurement of inflammatory mediators in lung tissue (follow-onanalysis). Right lung lobes (all remaining lobes) isolated forquantification of inflammatory mediators were snap frozen for subsequentmeasurement of IFN-gamma, IL-1 alpha, IL-1 beta, CXCL1, CCL3, CXCL2,CCL5, IL-17A, TNF-alpha, IL-17F, IL-23 and IL-33 by commerciallyavailable multiplex assay (Merck-Millipore). Analysis was performedaccording to the manufacturer's instructions.

Measurement of antibody levels in serum and BAL fluid (follow-onanalysis). House-dust-mite (HDM) specific IgG1 and IgG2a antibodyproduction were measured in the BAL and serum by ELISA assay.

Isolation of lung and histological analysis (follow-on analysis). Leftlung lobes were fixed in formalin followed by embedment in paraffin,sectioning, and staining with hematoxylin and eosin and PAS. Subsequenthistological scoring was performed blinded as followed: Five randomfields of view per sample were scored for inflammation (peribronchialinfiltration and perivascular infiltration) and mucus production.Inflammatory infiltration was scored with the following grading system:

0—normal

1—mild inflammatory infiltrates

2—moderate inflammatory infiltrates

3—marked inflammatory infiltrates

4—severe inflammatory infiltrates

5—very severe inflammatory infiltrates

Results and Analysis

The results of the experiments are shown in FIGS. 47-64.

Further analysis of antibody levels revealed that the efficacy ofbacterial strain 751 was also reflected in reduced HDM-specific IgG1levels in the BAL fluid and serum (see FIGS. 47 and 49). Firmconclusions regarding an effect on IgG2a levels cannot be drawn.Overall, the data from the antibody analysis is suggestive of areduction related to an overall reduced inflammatory response, asopposed to a selective effect on antibody isotype switching.

Histological analysis supported the differential cell counts from theBAL fluid, showing a reduced cellular infiltrate in mice treated withStrain 751 (see FIGS. 51-53).

In relation to cytokine levels, as for Example 4, the spread of data andthe error bars for each treatment are large, and the positive andnegative controls do not appear to have behaved as necessarily would beexpected. It is also possible that the mechanism of action involvesinfluencing earlier cytokine responses that were no longer detectable onday 4 post the final HDM airway challenge. Some care should be takenwhen interpreting the cytokine data in the current study, due to thevariability in the levels detected. This variability could in part beexplained by the fact that the lung tissue was separated for thedifferent analyses, and thus one lung lobe might not have been fullyrepresentative or comparable to the same lobe in other mice due topatchy distribution of the inflammation. Despite this variability, aclear anti-inflammatory effect on cytokine levels for strain 751 wasshown, and the positive control anti-IL-17 Ab generally behaved asexpected.

With the above caveats, the data in FIGS. 56, 58, 59, 61 and 63 suggestthat treatment with the bacterial strains of the invention, and inparticular strain 751 may achieve a reduction in the levels of IL-1b,IFNg, RANTES, MIP-1a and KC (the mouse orthologue of human IL-8), whichmay be indicative of a mechanism of action related to influences onchemokine release (and thus recruitment of cells) by stromal or innateimmune cells. These cytokines are part of the Th17 pathway. Taking thisdataset together, a clear conclusion can be drawn that Strain 751 washighly effective at protecting mice against inflammation in this mousemodel of severe neutrophilic asthma.

Example 6—Efficacy of Bacterial Inocula in a Mouse Model of MultipleSclerosis

Summary

Mice were administered with compositions comprising bacterial strainsaccording to the invention and the mice were subsequently immunised withmyelin oligodendrocyte glycoprotein to induce experimental autoimmuneencephalomyelitis (EAE). EAE is the most commonly used experimentalmodel for human multiple sclerosis. The compositions of the inventionwere found to have a striking effect on disease incidence and diseaseseverity.

Strain

751: bacterium deposited under accession number NCIMB 42380

Study Design

Groups:

1. Negative control group. Treatment with vehicle control (per oral).

4. Treatment with therapeutic bacteria inoculum strain 751 (per oral).

9. Positive control group. Treatment with Dexamethasone (i.p.).

10. Untreated Control Group.

Number of mice per group=10

Days −14 to day 27: Daily administration of vehicle control per oral(Group 1).

Days −14 to day 27: Daily administration of therapeutic bacteriainoculum per oral (Group 4).

Days 0-28: administration of Dexamethasone (i.p.) three times a week(Group 9)

Day 0: MOG35-55 (myelin oligodendrocyte glycoprotein—2 mg/ml) and CFA (2mg/ml MTB) were mixed 1:1 resulting in 1 mg/ml solutions. 100 d of thepeptide-CFA mixture was injected subcutaneously into each hind leg.Administration of pertussis toxin intraperitoneally (300 ng).Day 1: Administration of pertussis toxin intraperitoneally (300 ng).Days 7-onwards: Measurement of disease incidence and weight three timesa week.Endpoints and Analysis

Mice were analysed for disease incidence and disease severity threetimes a week. Scoring was performed blind. Disease severity was assessedusing a clinical score ranging from 0 to 5, with 5 indicating a deadmouse (see clinical scoring system below).

Monitoring

On the indicated days mice were weighed and observed for diseaseactivity score and disease incidence.

Disease activity score observations:

0—No obvious changes in motor function compared to non-immunized mice.

0.5—Tip of tail is limp.

1.0—Limp tail.

1.5—Limp tail and hind leg inhibition.

2.0—Limp tail and weakness of hind legs.

-   -   OR—There are obvious signs of head tilting when the walk is        observed. The balance is poor.        2.5—Limp tail and dragging of hind legs.    -   OR—There is a strong head tilt that causes the mouse to        occasionally fall over.        3.0—Limp tail and complete paralysis of hind legs.        3.5—Limp tail and complete paralysis of hind legs.    -   In addition to: Mouse is moving around the cage, but when placed        on its side, is unable to right itself.    -   Hind legs are together on one side of body.        4.0—Limp tail, complete hind leg and partial front leg        paralysis.    -   Mouse is minimally moving around the cage but appears alert and        feeding        4.5—Complete hind and partial front leg paralysis, no movement        around the cage.    -   Mouse is immediately euthanized and removed from cage.        5.0 Mouse is euthanized due to severe paralysis.

When an animal has equal or greater disease activity score of 1, it isconsidered to have a positive disease incidence score.

Results

The results of the study are shown in FIGS. 71 and 72.

Disease induction in the negative control groups was successful withhigh scores shown by the vehicle control and the untreated control. Theeffect of treatment with strain 751 was striking and the mice treatedwith strain 751 exhibited notably reduced disease incidence and diseaseseverity. Indeed, the reduction in disease incidence and diseaseseverity was comparable to the positive control group. These dataindicate the strain 751 may be useful for treating or preventingmultiple sclerosis.

Example 7—Efficacy of Bacterial Inocula in Mouse Models of Cancer

Summary

This study tested the efficacy of compositions comprising bacterialstrains according to the invention in four tumor models.

Materials Test substance—Bacterial strain #MRX004 (strain 751).

Reference substance—Anti-CTLA-4 antibody (clone: 9H10, catalog: BE0131,isotype: Syrian Hamster IgG1, Bioxcell).

Test and reference substances vehicles—Bacterial culture medium (Yeastextract, Casitone, Fatty Acid medium (YCFA)). Each day of injection tomice, antibody was diluted with PBS (ref: BE14-516F, Lonza, France).

Treatment doses—Bacteria: 2×10⁸ in 200 μL. The a-CTLA-4 was injected at10 mg/kg/inj. Anti-CTLA-4 was administered at a dose volume of 10mL/kg/adm (i.e. for one mouse weighing 20 g, 200 μL of test substancewill be administered) according to the most recent body weight of mice.

Routes of administration—Bacterial inoculum was administered by oralgavage (per os, PO) via a cannula. Cannulas were decontaminated everyday. Anti-CTLA-4 was injected into the peritoneal cavity of mice(Intraperitoneally, IP).

Culture conditions of bacterial strain—The culture conditions for thebacterial strain were as follows:

-   -   Pipette 10 mL of YCFA (from the prepared 10 mL E&O lab bottles)        into Hungate tubes    -   Seal the tubes and flush with CO₂ using a syringe input and        exhaust system    -   Autoclave the Hungate tubes    -   When cooled, inoculate the Hungate tubes with 1 mL of the        glycerol stocks    -   Place the tubes in a static 37° C. incubator for about 16 hours.    -   The following day, take 1 mL of this subculture and inoculate 10        mL of YCFA (pre-warmed flushed Hungate tubes again, all in        duplicate)    -   Place them in a static 37° C. incubator for 5 to 6 h        Cancer Cell Line and Culture Conditions—

The cell lines that were used are detailed in the table below:

Cell line Type Mouse strain Origin EMT-6 Breast carcinoma BALB/c ATCCLL/2 (LLC1) Lung carcinoma C57BL/6 ATCC CRL1642 Hepa 1-6 HepatocellularC57BL/6 IPSEN carcinoma INNOVATION

The EMT-6 cell line was established from a transplantable murine mammarycarcinoma that arose in a BALB/cCRGL mouse after implantation of ahyperplastic mammary alveolar nodule [73].

The LL/2 (LLC1) cell line was established from the lung of a C57BL mousebearing a tumor resulting from an implantation of primary Lewis lungcarcinoma [74].

The Hepa 1-6 cell line is a derivative of the BW7756 mouse hepatoma thatarose in a C57/L mouse [75].

Cell culture conditions—All cell lines were grown as monolayer at 37° C.in a humidified atmosphere (5% CO₂, 95% air). The culture medium andsupplement are indicated in the table below:

Cell line Culture medium Supplement EMT6 RPMI 1640 containing 2 mM 10%fetal bovine serum L-glutamine (ref: BE12-702F, Lonza) (ref: #3302,Lonza) LL/2 RPMI 1640 containing 2 mM 10% fetal bovine serum (LLC1)L-glutamine (ref: BE12-702F, Lonza) (ref: #3302, Lonza) Hepa 1-6 DMEM(ref: 11960-044, Gibco) 10% fetal bovine serum (ref: #3302, Lonza) 2 mML-Glutamine penicillin-streptomycin (Sigma G-6784)

For experimental use, adherent tumor cells were detached from theculture flask by a 5 minute treatment with trypsin-versene (ref:BE17-161E, Lonza), in Hanks' medium without calcium or magnesium (ref:BE10-543F, Lonza) and neutralized by addition of complete culturemedium. The cells were counted in a hemocytometer and their viabilitywill be assessed by 0.25% trypan blue exclusion assay.

Use of Animals—

Healthy female Balb/C (BALB/cByJ) mice, of matching weight and age, wereobtained from CHARLES RIVER (L'Arbresles) for the EMT6 modelexperiments.

Healthy female C57BL/6 (C57BL6J) mice, of matching weight and age, wereobtained from CHARLES RIVER (L'Arbresles) for the LL/2(LLC1) and theHepa1-6 model experiments.

Animals were maintained in SPF health status according to the FELASAguidelines, and animal housing and experimental procedures according tothe French and European Regulations and NRC Guide for the Care and Useof Laboratory Animals were followed [76,77]. Animals were maintained inhousing rooms under controlled environmental conditions: Temperature:22±2° C., Humidity 55±10%, Photoperiod (12 h light/12 h dark), HEPAfiltered air, 15 air exchanges per hour with no recirculation. Animalenclosures were provided with sterile and adequate space with beddingmaterial, food and water, environmental and social enrichment (grouphousing) as described: 900 cm² cages (ref: green, Tecniplast) inventilated racks, Epicea bedding (SAFE), 10 kGy Irradiated diet (A04-10,SAFE), Complete food for immuno-competent rodents—R/M-H Extrudate, waterfrom water bottles.

Experimental Design and Treatments

Antitumor Activity, EMT6 Model

Treatment schedule—The start of first dosing was considered as DO. OnDO, non-engrafted mice were randomized according to their individualbody weight into groups of 9/8 using Vivo Manager® software(Biosystemes, Couternon, France). On DO, the mice received vehicle(culture medium) or bacterial strain. On D14, all mice were engraftedwith EMT-6 tumor cells as described below. On D24, mice from thepositive control group received anti-CTLA-4 antibody treatments.

The treatment schedule is summarized in the table below:

No. Treatment Group Animals Treatment Dose Route Schedule 1 8 Untreated— — — 2 8 Vehicle (media) — PO Q1D × 42 3 9 Bacterial strain #1 2 × 108PO Q1D × 42 (MRX004) bacteria 4 8 Anti-CTLA4 10 mg/kg IP TW × 2

The monitoring of animals was performed as described below.

Induction of EMT6 tumors in animals—On D14, tumors were induced bysubcutaneous injection of 1×10⁶ EMT-6 cells in 200 μL RPMI 1640 into theright flank of mice.

Euthanasia—Each mouse was euthanized when it reached a humane endpointas described below, or after a maximum of 6 weeks post start of dosing.

Antitumor Activity, LL/2 (LLC1) Model

Treatment schedule—The start of first dosing was considered as DO. OnDO, non-engrafted mice were randomized according to their individualbody weight into 7 groups of 9/8 using Vivo Manager® software(Biosystemes, Couternon, France). On DO, the mice will received vehicle(culture medium) or bacterial strain. On D14, all mice were engraftedwith LL/2 tumor cells as described below. On D27, mice from the positivecontrol group received anti-CTLA-4 antibody treatments.

The treatment schedule is summarized in the table below:

No. Treatment Group Animals Treatment Dose Route Schedule 1 8 Untreated— — — 2 9 Vehicle (media) — PO Q1D × 42 3 9 Bacterial strain #1 2 × 10⁸PO Q1D × 42 (MRX004) bacteria 4 8 Anti-CTLA4 10 mg/kg IP TW × 2

The monitoring of animals was performed as described below.

Induction of LL/2 (LLC1) tumors in animals—On D14, tumors were inducedby subcutaneous injection of 1×10⁶ LL/2 (LLC1) cells in 200 μL RPMI 1640into the right flank of mice.

Euthanasia—Each mouse was euthanized when it reached a humane endpointas described below, or after a maximum of 6 weeks post start of dosing.

Antitumor Activity, Hepa1-6 Model

Treatment schedule—The start of first dosing was considered as DO. OnDO, non-engrafted mice were randomized according to their individualbody weight into 7 groups of 9 using Vivo Manager® software(Biosystemes, Couternon, France). On DO, the mice received vehicle(culture medium) or bacterial strain. On D14, all mice were engraftedwith Hepa 1-6 tumor cells as described below. On D16, mice from thepositive control group received anti-CTLA-4 antibody treatments.

The treatment schedule is summarized in the table below:

No. Treatment Group Animals Treatment Dose Route Schedule 1 9 Untreated— — — 2 9 Vehicle (media) — PO Q1D × 42 4 9 Bacterial strain #2 2 × 10⁸PO Q1D × 42 (MRX004) bacteria 7 9 Anti-CTLA4 10 mg/kg IP TW × 2

The monitoring of animals was performed as described below.

Orthotopic induction of Hepa 1-6 tumor cells in animals by intrasplenicinjection—On D14, one million (1×10⁶) Hepa 1-6 tumor cells in 50 μL RPMI1640 medium were transplanted via intrasplenic injection into mice.Briefly, a small left subcostal flank incision was made and the spleenwas exteriorized. The spleen was exposed on a sterile gauze pad, andinjected under visual control with the cell suspension with a 27-gaugeneedle. After the cell inoculation, the spleen was excised.

Euthanasia—Each mouse was euthanized when it reached a humane endpointas described in section below, or after a maximum of 6 weeks post startof dosing.

Evaluation of tumor burden at euthanasia—At the time of termination,livers were collected and weighed.

Animal Monitoring

Clinical monitoring—The length and width of the tumor was measured twicea week with callipers and the volume of the tumor was estimated by thisformula [78]:

${{Tumor}\mspace{14mu}{volume}} = \frac{{width}^{2} \times {length}}{2}$

Humane endpoints [79]: Signs of pain, suffering or distress: painposture, pain face mask, behaviour; Tumor exceeding 10% of normal bodyweight, but non-exceeding 2000 mm³; Tumors interfering with ambulationor nutrition; Ulcerated tumor or tissue erosion; 20% body weight lossremaining for 3 consecutive days; Poor body condition, emaciation,cachexia, dehydration; Prolonged absence of voluntary responses toexternal stimuli; Rapid laboured breathing, anaemia, significantbleeding; Neurologic signs: circling, convulsion, paralysis; Sustaineddecrease in body temperature; Abdominal distension.

Anaesthesia—Isoflurane gas anesthesia were used for all procedures:surgery or tumor inoculation, i.v. injections, blood collection.Ketamine and Xylazine anesthesia were used for stereotaxia surgicalprocedure.

Analgesia—Carprofen or multimodal carprofen/buprenorphine analgesiaprotocol were adapted to the severity of surgical procedure.Non-pharmacological care was provided for all painful procedures.Additionally, pharmacological care not interfering with studies (topictreatment) were provided at the recommendation of the attendingveterinarian.

Euthanasia—Euthanasia of animals was performed by gas anesthesiaover-dosage (Isoflurane) followed by cervical dislocation orexsanguination.

Results

Antitumor Activity, EMT6 Model

The results are shown in FIG. 73. Treatment with the bacterial strain ofthe invention led to a clear reduction in tumour volume relative to boththe negative controls. The positive control also led to a reduction intumour volume, as would be expected.

Antitumor Activity, LL/2 (LLC1) Model

The results are shown in FIG. 74. The negative and positive controls donot appear as would be expected, because tumour volume was greater inthe mice treated with the positive control than in the negative controlgroups. Nevertheless, tumour volume in the mice treated with thebacterial strain of the invention was comparable to the positive controlgroup, which is consistent with a useful therapeutic effect.

Antitumor Activity, Hepa1-6 Model

The results are shown in FIG. 75. The untreated negative control doesnot appear as would be expected, because liver weight was lower in thisgroup than the other groups. However, the vehicle negative control andthe positive control groups both appear as would be expected, becausemice treated with vehicle alone had larger livers than mice treated withanti-CTLA4 antibodies, reflecting a greater tumour burden in the vehiclenegative control group. Treatment with the bacterial strain of theinvention led to a clear reduction in liver weight (and therefore tumourburden) relative to the mice in the vehicle negative control group.

These data indicate that strain 751/MRX004 may be useful for treating orpreventing cancer, and in particular for reducing tumour volume inbreast, lung and liver cancers.

Example 8—Attachment to Human Cells in YCFA Medium

Summary

The level of binding of strain 751 and a number of Bifidobacterium brevestrains to human cells was determined at 3 distinct time points in YCFAmedium. The bacteria attached to the human cells were resuspended inmedium and the optical density of the medium was then analysed—thehigher the optical density, the higher the number of bacterial cells andthus, the higher the level of binding of the bacterial cells to humancells. The 751 strain was found to display reduced attachment to humancells compared to the Bifidobacterium breve reference strains.

Results and Analysis

The results of the experiment are shown in FIG. 76.

As shown in FIG. 76, the Bifidobacterium breve strains show a high levelof attachment to human cells at all time points. On the other hand, the751 strain has a drastically reduced level of attachment to human cells.Therefore, the low adherence to human cells of strain 751 may increasethe beneficial effect of the compositions of the invention on the IL-17or the Th17 pathway and on diseases mediated by IL-17 or the Th17pathway.

Example 9—Assay Detecting the Production of Exopolysaccharides

Summary

The level of exopolysaccharide (EPS) production by the bacterial strainof the invention (751) and a number of Bifidobacterium breve strains wasanalysed at 37° C. for 48 hours and at 30° C. for 72 hours. EPSs arepolysaccharides produced by certain bacteria which bind to the outsidesurface of the bacterial cell. The level of EPSs on the surface ofbacteria can be determined using a Congo Red assay which binds to thepolysaccharides. A higher intensity of Congo Red absorbance indicates ahigher concentration of EPSs on the surface of the bacteria. Thebacterial strain of the invention was found to produce and bind moreEPSs than the Bifidobacterium breve strains.

Results and Analysis

The results of this experiment are shown in FIG. 77.

As shown in FIG. 77, the bacterial strain of the invention showed agreater Congo Red absorbance than the Bifidobacterium breve strains atboth temperatures and time-points. Therefore, the strain of theinvention displays greater EPS production and a greater level ofextracellular bound EPSs. As the EPSs enable bacteria to bind to mucusand epithelial cells, the bacterial strain of the invention may beuseful for competing with pathogenic cells for binding sites onepithelial cells and within mucus membranes. Thus, the bacterial strainof the invention may be useful for modulating the microbiome andtreating a number of diseases associated with the microbiome.

Example 10—Stability Testing

A composition described herein containing at least one bacterial straindescribed herein is stored in a sealed container at 25° C. or 4° C. andthe container is placed in an atmosphere having 30%, 40%, 50%, 60%, 70%,75%, 80%, 90% or 95% relative humidity. After 1 month, 2 months, 3months, 6 months, 1 year, 1.5 years, 2 years, 2.5 years or 3 years, atleast 50%, 60%, 70%, 80% or 90% of the bacterial strain shall remain asmeasured in colony forming units determined by standard protocols.

Example 11—MRX004 Bound and Released Exopolysaccharide Production Assay

For EPS extraction, MRX004 was cultured in 10 ml YCFA until it reachedlate exponential phase, when bacterial cells and supernatants wereseparated by centrifugation. Cells were washed once with PBS to removeany remaining culture medium. Secreted or released EPS (EPS-R) wasprecipitated from culture supernatants by treatment with ice-cold 100%ethanol (overnight at 4° C. with gentle agitation). To extract capsularor bound EPS (EPS-B), cells were incubated with 0.05 M EDTA (overnightat 4° C. with gentle agitation), and supernatants from this treatmentwere collected and subsequently treated with 100% ice-cold ethanol(overnight at 4° C. with gentle agitation) to precipitate EPS-B.Precipitated EPS-B and EPS-R were pelleted by centrifugation and wereallowed to dry briefly in a laminar hood before they were resuspended inenough sterile Ultrapure water to achieve a uniform solution. To furtherpurify samples, they were dialysed against sterile Ultrapure water at aratio of 1:100 for 48 hours with 3 buffer changes. EPS-B and EPS-R werequantified using the phenol-sulphuric acid method using glucose asstandard. Using this assay, MRX004 was found to produce a greater amountof EPS-R (115 μg) than EPS-B (17 μg) (FIG. 78).

Example 12—Attachment Assay of MRX004 to Caco-2 Cells

Attachment of MRX004 to host cells was analysed using an in vitroco-culture assay with Caco-2 intestinal epithelial cells. Caco-2 cellswere seeded at a density of 1×10 Bacteria were cultured in 10 ml YCFAuntil they reached late exponential phase, when they were pelleted,washed twice with PBS, and resuspended in antibiotic-free cell culturemedium. Bacterial density was adjusted to obtain an approximatemultiplicity of infection (MOI) of 10:1 (which was confirmed by platingon YCFA agar using the WASP standard protocol), and MRX004 wasco-incubated with Caco-2 cells in anaerobic conditions at 37° C. for 2hours. Medium was subsequently removed, and unbound bacteria wereremoved by washing Caco-2 cells three times with PBS. Bacteria-boundCaco-2 cells were lysed and removed from the vessel using treatment with0.1% Triton X-100, and 50 μl volumes of diluted lysate were plated inYCFA agar using the WASP. Attachment was calculated by counting thenumbers of bacteria recovered from lysate and expressing this as apercentage of total bacteria. MRX004 was found to display low-leveladherence (0.3% of total culture) to Caco-2 cells (FIG. 79).

Example 13—Characterisation of Enzymatic Activity

The Analytical Profile Index (API®) test system consists of strips thatcontain miniaturised biochemical tests that assay for enzymatic activityin bacterial species. MRX004 (strain 751, the bacterium deposited underaccession number NCIMB 42380) was characterised using two API testsystems: Rapid ID 32A—This system is designed specifically for anaerobicspecies and encompasses tests for carbohydrate, amino acid and nitratemetabolism as well as alkaline phosphatase activity; and API® 50 CH—Thissystem tests for the fermentation of 49 carbohydrate sources, and can beutilised in conjunction with API® CHL Medium for analysis of anaerobicspecies.

Rapid ID 32A testing was carried out on bacterial colonies as permanufacturer's instructions. Briefly, bacteria were cultured on YCFAagar for 24 hours at 37° C. in an anaerobic workstation. Colonies wereremoved from plates using a sterile 5 μl inoculating loop andresuspended in a 2 ml ampoule of API® Suspension Medium until a densityroughly equivalent to that of McFarland standard No. 4 was achieved.Fifty-five microlitres of bacterial suspension was added to each cupuleon a Rapid ID 32A strip, and the urease test was overlayed with twodrops of mineral oil. Strips were covered with a plastic lid andincubated aerobically at 37° C. for 4 hours, following which the bottomrow of cupules were developed using the following reagents: NIT: 1 dropeach of NIT1 and NIT2; IND: 1 drop of James reagent; all remainingcupules: 1 drop of FastBlue reagent. Strips were incubated at roomtemperature for 5 minutes, following which the colour of each cupule wasrecorded and assigned a value of negative, intermediate positive orpositive.

The results of the Rapid ID 32A analysis are shown in FIG. 80. MRX004tested positive for fermentation of several carbohydrate sources, namelyα-galactosidase and β-galactosidase, α-glucosidase and β-glucosidase,β-arabinose, mannose and raffinose, as well as the amino acids arginine,proline, phenylalanine, leucine, tyrosine, glycine and histidine.Interestingly, roles have been reported for some of these amino acids inasthma. For instance, increased plasma concentrations of phenylalanineand histidine have been reported to be associated with adverse effectsin asthma, including increased inflammation, histamine production andairway hyper-responsiveness. In addition, arginine metabolism isimplicated in asthma pathogenesis, as increased levels of the argininemetabolite L-omithine have been reported in paediatric patients, andadministration of arginine attenuated inflammation in an in vivo asthmamodel. Based on these reports, it is possible that amino acid metabolismby MRX004 may be involved in the anti-asthma effects of this strain.

Comparative Rapid ID 32A analysis was carried out between MRX004 andfour B. breve type strains, which are annotated in FIG. 80B as Bif Ref 1(DSM 20091), Bif Ref 2 (DSM 20213), Bif Ref 6 (JCM 7017) and Bif Ref 7(UCC2003). This analysis demonstrated that MRX004 was the only straintested to ferment the polysaccharide raffinose, which may besignificant, because raffinose is involved in the production ofbacterial components such as exopolysaccharides, and raffinosefermentation can also reportedly confer effects on the host such asincreased caecal butyrate, increased gastrointestinal proliferation andweight loss.

API® 50 CH testing was carried out to further examine carbohydratemetabolism in MRX004. As per manufacturer's instructions, bacteria werecultured in 10 ml YCFA broth for 16-18 hours at 37° C. in an anaerobicworkstation. This culture was diluted in 10 ml API® CHL Medium so as toachieve a density roughly equivalent to McFarland standard No. 2, and110 μl of this mixture was used to inoculate each cupule on a set ofAPI® 50 CH test strips. Test strips were incubated in a humidifiedincubation box at 37° C. in an anaerobic workstation for 48 hours,following which the colour of each cupule was recorded and assigned avalue of negative, intermediate positive, positive or doubtful.

Using API® 50, MRX004 tested positive for utilisation of the followingcarbohydrate sources: amidon (starch), amygdalin, arbutin, cellobiose,esculin, galactose, gentiobiose, glucose, glycogen, fructose, fucose,lactose, maltose, mannose, mannitol, melibiose, melezitose, methylα-D-glucopyranoside, N-acetylglucosamine, ribose, saccharose (sucrose),salicin, sorbitol, trehalose, turanose and xylitol. These resultscorrelated with those obtained for Rapid ID 32A testing in that MRX004demonstrated fermentation of galactose, glucose, mannose and raffinosein both test systems. Interestingly, some MRX004 carbohydratesubstrates, namely galactose and fructose, may be implicated in themechanism of action of this strain, based on their reported effects inthe literature. Galactose α-1,3-galactose derived from meat sources is aknown allergen and causative agent of anaphylaxis, and intake levels ofdietary fructose are correlated with increased asthma severity. Takentogether both sets of API® data for MRX004 suggest that the metabolismof this strain may play a role in its anti-asthma effects.

Example 14—Genome Analysis

A comparison of the genome content of strain MRX004 and the referencestrains of B. breve, 1, 2, 6 and 7 was carried out using blastn as partof the BLAST+2.3.0 suite of programs. A maximum E-value cut-off score of10E-5 was employed throughout the analysis.

333 genes were identified (Table 1) that are present in the genome ofstrain MRX004 but are absent from the B. breve reference strains 1 (DSM20091), 2 (DSM 20213), 6 (JCM 7017) and 7 (UCC2003). Many of the geneslisted in Table 1 are frequently observed as being hypervariable amongB. breve strains [80]. As expected, the regions of variability includegenes that code for proteins involved in carbohydrate metabolism andtransport, phage-associated genes, mobile elements, as well as 173 genespredicted to encode proteins or genes of unknown function.

Genes that are present in MRX004 but absent from B. breve referencestrains 1, 2, 6 and 7 are listed in Table 1. Genes that are nothighlighted are absent in more than one of the four reference strains.The large number of genes that are present in MRX004 but are not presentin numerous B. breve reference strains suggests that MRX004 is distinctfrom and/or distinguishable from these known B. breve strains. Geneshighlighted with single underlining are present in MRX004 but absent inB. breve reference strain 1. Genes highlighted with double underliningand in bold are present in MRX004 but absent in B. breve referencestrain 2. Genes highlighted with italics are present in MRX004 butabsent in B. breve reference strain 6. A maximum E-value cut-off scoreof 10E-5 was employed for the blastn analysis.

TABLE 1 4DBb_0021c Multiple sugar ABC transporter, substrate-bindingprotein 4DBb_0023 Probable LacI-type transcriptional regulator 4DBb_0024Sucrose-6-phosphate hydrolase (EC 3.2.1.B3)

4DBb_0036c Hypothetical protein 4DBb_0038c MSM (multiple sugarmetabolism) operon regulatory protein 4DBb_0119c Hypothetical protein4DBb_0120c Hypothetical protein 4DBb_0187 Hypothetical protein 4DBb_0188Hypothetical protein 4DBb_0203c Cell division protein FtsL 4DBb_0204cHypothetical protein 4DBb_0205c Hypothetical protein 4DBb_0206cTranscriptional regulator, Hx1R family 4DBb_0207 Rrf2-linked NADH-flavinreductase 4DBb_0208 ATP-dependent DNA helicase RecG-related protein4DBb_0209c Hypothetical protein 4DBb_0210 Putative transporter 4DBb_0211Omega-3 polyunsaturated fatty acid synthase subunit, PfaA 4DBb_0212 TypeI polyketide synthase 4DBb_0213c Hypothetical protein 4DBb_0214cHypothetical protein 4DBb_0215 Hypothetical protein 4DBb_0216c Conservedhypothetical protein 4DBb_0218c Hypothetical protein 4DBb_0219cDNA-cytosine methyltransferase 4DBb_0220c Hypothetical protein4DBb_0221c Hypothetical protein 4DBb_0222c Hypothetical protein4DBb_0223c Integrase 4DBb_0256 Hypothetical protein 4DBb_0257c LacI-typetranscriptional regulator 4DBb_0258 Putative glycosyl hydrolase ofunknown function (DUF1680) 4DBb_0284 Transcriptional regulator, AraCfamily 4DBb_0285 N-Acetyl-D-glucosamine ABC transport system,sugar-binding protein 4DBb_0286 Sugar ABC transporter permease 4DBb_0287N-Acetyl-D-glucosamine ABC transport system, permease protein 24DBb_0288 Alpha-galactosidase (EC 3.2.1.22) 4DBb_0329c ATPase componentBioM of energizing module of biotin ECF transporter 4DBb_0330 Majorfacilitator superfamily MFS_1 4DBb_0368 GMP synthase [glutamine-hydrolyzing] (EC 6.3.5.2) 4DBb_0369c Mu-like prophage protein gp29

4DBb_0419 Hypothetical protein 4DBb_0421 Glycosyltransferase SypP4DBb_0422 Capsular polysaccharide biosynthesis protein 4DBb_0423Hypothetical protein 4DBb_0424 Glycosyltransferase 4DBb_0425 Membraneprotein involved in the export of O-antigen, teichoic acid lipoteichoicacids 4DBb_0426 Glycosyltransferase (EC 2.4.1.-) 4DBb_04272-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene- 1-carboxylic-acidsynthase (EC 2.2.1.9) 4DBb_0428 Hypothetical protein 4DBb_0429 Mobileelement protein 4DBb_0430 Hypothetical protein 4DBb_0431c Hypotheticalprotein 4DBb_0432c Mobile element protein 4DBb_0433c Hypotheticalprotein 4DBb_0434c Hypothetical protein 4DBb_0435 Hypothetical protein4DBb_0436 Hypothetical protein 4DBb_0437c Hypothetical protein4DBb_0438c Mobile element protein 4DBb_0439c Hypothetical protein4DBb_0440c Mobile element protein 4DBb_0518c PIN domain protein4DBb_0519c Hypothetical protein 4DBb_0555c Hypothetical protein4DBb_0556c Hypothetical protein 4DBb_0557c LSU ribosomal protein L31p @LSU ribosomal protein L31p, zinc-independent 4DBb_0558c SSU ribosomalprotein S14p (S29e) @ SSU ribosomal protein S14p (S29e),zinc-independent 4DBb_0559c LSU ribosomal protein L33p @ LSU ribosomalprotein L33p, zinc-independent 4DBb_0560c Hypothetical protein 4DBb_0561Hypothetical protein

4DBb_0660 Mobile element protein 4DBb_0662 Mobile element protein4DBb_0663c Neuraminidase NanP 4DBb_0664 Hypothetical protein 4DBb_0665Hypothetical protein 4DBb_0666 Mobile element protein 4DBb_0667 Mobileelement protein 4DBb_0668 Mobile element protein 4DBb_0718 Predictedbiotin regulatory protein BioR (GntR family) 4DBb_0719 Hypotheticalprotein 4DBb_0720 Hypothetical protein 4DBb_0778 Hypothetical protein4DBb_0789c Mobile element protein 4DBb_0790c Mobile element protein4DBb_0837c Possible conserved integral membrane protein. 4DBb_0840Macrolide-efflux protein 4DBb_0866 Cation-transporting ATPase, E1-E2family 4DBb_0867 Hypothetical protein 4DBb_0872 Hypothetical protein4DBb_0879c Transcriptional regulator, Cro/CI family 4DBb_0880c Integralmembrane protein 4DBb_0946 MFS general substrate transporter 4DBb_0947cPutative membrane protein 4DBb_0948c Mobile element protein 4DBb_0952cHypothetical protein 4DBb_0953 Hypothetical protein 4DBb_0954cHypothetical protein 4DBb_0955c Hypothetical protein 4DBb_0956cPredicted permeases 4DBb_0957 Narrowly conserved hypothetical protein4DBb_0958c Conserved hypothetical protein 4DBb_0986cHypothetical protein 4DBb_0987c Duplicated ATPase component BL0693 ofenergizing module of predicted ECF transporter 4DBb_0988cTransmembrane component BL0694 ofenergizing module of predicted ECF transporter 4DBb_1009c Phage holin4DBb_1010c Membrane-bound lytic murein transglycosylase D precursor (EC3.2.1.-) 4DBb_1011c Hypothetical protein 4DBb_1012 Hypothetical protein4DBb_1013c Hypothetical protein 4DBb_1014c Hypothetical protein4DBb_1015c Hypothetical protein 4DBb_1016c Hypothetical protein4DBb_1017c Phage tail protein 4DBb_1018c Phage tail length tape-measureprotein 4DBb_1019c Hypothetical protein 4DBb_1020c Hypothetical protein4DBb_1021c Hypothetical protein 4DBb_1022c Hypothetical protein4DBb_1023c hypothetical protein 4DBb_1024c Hypothetical protein4DBb_1025c Hypothetical protein 4DBb_1026c Hypothetical protein4DBb_1027c Hypothetical protein 4DBb_1028c Hypothetical protein4DBb_1029c Hypothetical protein 4DBb_1030c Phage protein 4DBb_1031cPhage terminase, large subunit #Pham2 4DBb_1032c Phage terminase, largesubunit 4DBb_1033c hypothetical protein 4DBb_1034 hypothetical protein4DBb_1035 hypothetical protein 4DBb_1036c FIG00424913: hypotheticalprotein 4DBb_1037c hypothetical protein 4DBb_1038c hypothetical protein4DBb_1039c hypothetical protein 4DBb_1040c hypothetical protein4DBb_1041c hypothetical protein 4DBb_1042c hypothetical protein4DBb_1043c hypothetical protein 4DBb_1044c hypothetical protein4DBb_1045c hypothetical protein 4DBb_1046c hypothetical protein4DBb_1047c Chromosome (plasmid) partitioning protein ParB 4DBb_1048chypothetical protein 4DBb_1049c hypothetical protein 4DBb_1050cSingle-stranded DNA-binding protein 4DBb_1051c hypothetical protein4DBb_1052c hypothetical protein 4DBb_1053c hypothetical protein4DBb_1054c hypothetical protein 4DBb_1055c hypothetical protein4DBb_1056 hypothetical protein 4DBb_1057c hypothetical protein4DBb_1058c hypothetical protein 4DBb_1059 hypothetical protein 4DBb_1060hypothetical protein 4DBb_1061 hypothetical protein 4DBb_1062chypothetical protein 4DBb_1063c hypothetical protein 4DBb_1064 putativephage integrase 4DBb_1113 Permeases of the major facilitator superfamily4DBb_1142 hypothetical protein 4DBb_1143c hypothetical protein 4DBb_1172Integrase 4DBb_1173c hypothetical protein 4DBb_1174c Narrowly conservedhypothetical protein 4DBb_1175 hypothetical protein 4DBb_1176chypothetical protein 4DBb_1177 ABC transporter, ATP-binding protein4DBb_1178 hypothetical protein 4DBb_1179 hypothetical protein 4DBb_1180hypothetical protein 4DBb_1181 hypothetical protein 4DBb_1182two-component system sensor kinase 4DBb_1183 hypothetical protein4DBb_1203c regulatory protein, LacI 4DBb_1204c FIG01131316: hypotheticalprotein 4DBb_1205c transport system permease 4DBb_1206c Predictedrhamnose oligosaccharide ABC transport system, permease component 24DBb_1207c extracellular solute-binding protein, family 1

4DBb_1213c hypothetical protein 4DBb_1214 Mobile element protein4DBb_1215 Mobile element protein 4DBb_1219 Hypothetical protein4DBb_1220c Hypothetical protein, 4DBb_1221c Protein of unknown functionDUF262 family 4DBb_1222c Hypothetical protein 4DBb_1223 Hypotheticalprotein 4DBb_1224 Mobile element protein 4DBb_1234c ABC-type sugartransport system, periplasmic component 4DBb_1235 Hypothetical protein4DBb_1328c Putative phage integrase 4DBb_1329 Hypothetical protein4DBb_1330 Hypothetical protein 4DBb_1331c Hypothetical protein 4DBb_1332Hypothetical protein 4DBb_1333 Hypothetical protein 4DBb_1334Hypothetical protein 4DBb_1335c Hypothetical protein 4DBb_1336c Negativeregulator of beta-lactamase expression 4DBb_1337c Hypothetical protein4DBb_1338c Hypothetical protein 4DBb_1339c Hypothetical protein4DBb_1340c Hypothetical protein 4DBb_1341c Hypothetical protein4DBb_1342c Hypothetical protein 4DBb_1343c Hypothetical protein4DBb_1344 Hypothetical protein 4DBb_1345c Phage tail fiber protein4DBb_1346c Hypothetical protein 4DBb_1347c Phage minor tail protein4DBb_1348c Hypothetical protein 4DBb_1349c Hypothetical protein4DBb_1350c Phage protein 4DBb_1351c Phage protein 4DBb_1352c Phageprotein 4DBb_1353c Phage protein 4DBb_1354c Hypothetical protein4DBb_1355c Hypothetical protein 4DBb_1356c Phage major capsid protein#Fam0025 #Pham164 4DBb_1357c Putative phage prohead protease 4DBb_1358cPhage portal protein 4DBb_1359c gp2, terminase 4DBb_1360c Hypotheticalprotein 4DBb_1361c Hypothetical protein 4DBb_1362c Hypothetical protein4DBb_1363c Hypothetical protein 4DBb_1364c Hypothetical protein4DBb_1365c Hypothetical protein 4DBb_1366c Hypothetical protein4DBb_1367c Hypothetical protein 4DBb_1368c Hypothetical protein4DBb_1369c Hypothetical protein 4DBb_1370c Hypothetical protein4DBb_1371c Hypothetical protein 4DBb_1372c Single-stranded DNA-bindingprotein 4DBb_1373c Hypothetical protein 4DBb_1374c Recombinational DNArepair protein RecT (prophage associated) 4DBb_1375c phage-relatedprotein 4DBb_1376c Hypothetical protein 4DBb_1377c Hypothetical protein4DBb_1378c Hypothetical protein 4DBb_1379c Hypothetical protein4DBb_1380c Methyltransferase (EC 2.1.1.-) 4DBb_1381c Hypotheticalprotein 4DBb_1382c Hypothetical protein 4DBb_1383c Hypothetical protein4DBb_1384 Hypothetical protein 4DBb_1385 Hypothetical protein 4DBb_1386Hypothetical protein 4DBb_1387 Hypothetical protein 4DBb_1388Hypothetical protein 4DBb_1456 Sucrose permease, major facilitatorsuperfamily 4DBb_1486c Esterase/lipase 4DBb_1487c Glucose/mannose: H+symporter GlcP 4DBb_1488c Two-component response regulator yesN

4DBb_1534c Hypothetical protein 4DBb_1535c Type Irestriction-modification system, restriction subunit R (EC 3.1.21.3)4DBb_1536c ATP-dependent DNA helicase recG (EC 3.6.1.-) 4DBb_1537c TypeI restriction-modification system, specificity subunit S (EC 3.1.21.3)4DBb_1538c Type I restriction-modification system, DNA-methyltransferase subunit M (EC 2.1.1.72) 4DBb_1539c Hypotheticalprotein 4DBb_1540 Type I restriction-modification system, specificitysubunit S (EC 3.1.21.3) 4DBb_1541 Integrase 4DBb_1542c Type Irestriction-modification system, specificity subunit S (EC 3.1.21.3)4DBb_1545c Ribose ABC transport system, high affinity permease RbsD (TC3.A.1.2.1) 4DBb_1546c Ribose ABC transport system, periplasmicribose-binding protein RbsB (TC 3.A.1.2.1) 4DBb_1547c Ribose ABCtransport system, permease protein RbsC (TC 3.A.1.2.1) 4DBb_1548c RiboseABC transport system, ATP-binding protein RbsA (TC 3.A.1.2.1) 4DBb_1550cHypothetical protein 4DBb_1551 Hypothetical protein 4DBb_1552c LacI-typetranscriptional regulator 4DBb_1553c Similar to tetracycline resistanceprotein 4DBb_1554c Ribokinase (EC 2.7.1.15) 4DBb_1555c NADH-dependentbutanol dehydrogenase A (EC 1.1.1.-) 4DBb_1556c Phosphoglycolatephosphatase (EC 3.1.3.18) 4DBb_1557c Inosine-undine preferringnucleoside hydrolase (EC 3.2.2.1) 4DBb_1558c Fructokinase (EC 2.7.1.4)4DBb_1559c Phosphoribosylanthranilate isomerase (EC 5.3.1.24) 4DBb_1560cATPase component STY3233 of energizing module of queuosine-regulated ECFtransporter 4DBb_1561c ATPase component of general energizing module ofECF transporters 4DBb_1562c Transmembrane component STY3231 ofenergizing module of queuosine-regulated ECF transporter 4DBb_1563cSubstrate-specific component STY3230 of queuosine-regulated ECFtransporter 4DBb_1564 Hypothetical sugar kinase in cluster withindigoidine synthase indA, PfkB family of kinases 4DBb_1569Transcriptional regulator, TetR family 4DBb_1570c Esterase/lipase4DBb_1571c Hypothetical protein 4DBb_1572c Hypothetical protein4DBb_1573 COG1309: Transcriptional regulator 4DBb_1574c Hypotheticalprotein 4DBb_1578c Melibiose carrier protein 4DBb_1579c Hypotheticalprotein 4DBb_1580c Transcriptional regulator, TetR family 4DBb_1581Hypothetical protein 4DBb_1582 Two-component response regulatorcolocalized with HrtAB transporter 4DBb_1583 Hypothetical protein4DBb_1584c Hypothetical protein 4DBb_1585 Sensor histidine kinase4DBb_1586 Mg(2+) transport ATPase protein C 4DBb_1587 Transcriptionalregulator, AbrB family 4DBb_1588 Hypothetical protein 4DBb_1620c3′-to-5′ oligoribonuclease (orn) 4DBb_1769c Transcriptional activatorMltR 4DBb_1770c Xylitol dehydrogenase (EC 1.1.1.9) 4DBb_1771cRibitol/Xylitol/Arabitol transporter, MFS superfamily 4DBb_1773cGlyoxalase family protein 4DBb_1774c Ribitol/Xylitol/Arabitoltransporter, MFS superfamily 4DBb_1775c Sorbitol dehydrogenase (EC1.1.1.14) 4DBb_1926c Membrane protein, related to Actinobacillus protein(1944168) 4DBb_1928c Glycosyl transferase, group 2 family protein4DBb_1929c Teichoic acid export ATP-binding protein TagH (EC 3.6.3.40)4DBb_1930c Rhamnose-containing polysacharide translocation permease4DBb_1934c Hypothetical protein 4DBb_1935 Possible glycosyltransferase4DBb_1936c Cell wall surface anchor family protein 4DBb_1937cD-alanyl-D-alanine carboxypeptidase (EC 3.4.16.4) 4DBb_1965 Hypotheticalprotein 4DBb_2010c Mobile element protein 4DBb_2011 Transcriptionalregulator, LacI family 4DBb_2012 Xylose ABC transporter, periplasmicxylose-binding protein XylF 4DBb_2013 Ribose ABC transport system,ATP-binding protein RbsA (TC 3.A.1.2.1) 4DBb_2014 Ribose ABC transportsystem, permease protein RbsC (TC 3.A.1.2.1) 4DBb_2015 Hypotheticalprotein 4DBb_2016 Mobile element protein 4DBb_2028c Beta-glucosidase (EC3.2.1.21)

Sequences (consensus 16S rRNA sequence for strain 751) SEQ ID NO: 1GGGACAGGCTCAGGATGAACGCCGGCGGCGTGCTTAACACATGCAAGTCGAACGGGATCCATCGGGCTTTGCCTGGTGGTGAGAGTGGCGAACGGGTGAGTAATGCGTGACCGACCTGCCCCATGCACCGGAATAGCTCCTGGAAACGGGTGGTAATGCCGGATGCTCCATCACACCGCATGGTGTGTTGGGAAAGCCTTTGCGGCATGGGATGGGGTCGCGTCCTATCAGCTTGATGGCGGGGTAACGGCCCACCATGGCTTCGACGGGTAGCCGGCCTGAGAGGGCGACCGGCCACATTGGGACTGAGATACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCGACGCCGCGTGAGGGATGGAGGCCTTCGGGTTGTAAACCTCTTTTGTTAGGGAGCAAGGCACTTTGTGTTGAGTGTACCTTTCGAATAAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCAAGCGTTATCCGGAATTATTGGGCGTAAAGGGCTCGTAGGCGGTTCGTCGCGTCCGGTGTGAAAGTCCATCGCTTAACGGTGGATCCGCGCCGGGTACGGGCGGGCTTGAGTGCGGTAGGGGAGACTGGAATTCCCGGTGTAACGGTGGAATGTGTAGATATCGGGAAGAACACCAATGGCGAAGGCAGGTCTCTGGGCCGTTACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGGTGGATGCTGGATGTGGGGCCCGTTCCACGGGTTCCGTGTCGGAGCTAACGCGTTAAGCATCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGAAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGCGGATTAATTCGATGCAACGCGAAGAACCTTACCTGGGCTTGACATGTTCCCGACGATCCCAGAGATGGGGTTTCCCTTCGGGGCGGGTTCACAGGTGGTGCATGGTCGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCGCCCCGTGTTGCCAGCGGATTGTGCCGGGAACTCACGGGGGACCGCCGGGGTTAACTCGGAGGAAGGTGGGGATGACGTCAGATCATCATGCCCCTTACGTCCAGGGCTTCACGCATGCTACAATGGCCGGTACAACGGGATGCGACAGCGCGAGCTGGAGCGGATCCCTGAAAACCGGTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGGCGGAGTCGCTAGTAATCGCGAATCAGCAACGTCGCGGTGAATGCGTTCCCGGGCCTTGTACACACCGCCCGTCAAGTCATGAAAGTGGGCAGCACCCGAAGCCGGTGGCCTAACC CCTGCGGGAGGGAGCCKC(strain 751 genome sequence)- SEQ ID NO: 2 see electronic sequence listing.

REFERENCES

-   [1] Spor et al. (2011) Nat Rev Microbiol. 9(4):279-90.-   [2] Eckburg et al. (2005) Science. 10; 308(5728):1635-8.-   [3] Macpherson et al. (2001) Microbes Infect. 3(12):1021-35-   [4] Macpherson et al. (2002) Cell Mol Life Sci. 59(12):2088-96.-   [5] Mazmanian et al. (2005) Cell 15; 122(1):107-18.-   [6] Frank et al. (2007) PNAS 104(34):13780-5.-   [7] Scanlan et al. (2006) J Clin Microbiol. 44(11):3980-8.-   [8] Kang et al. (2010) Inflamm Bowel Dis. 16(12):2034-42.-   [9] Machiels et al. (2013) Gut. 63(8):1275-83.-   [10] WO 2013/050792-   [11] WO 03/046580-   [12] WO 2013/008039-   [13] WO 2014/167338-   [14] Goldin and Gorbach (2008) Clin Infect Dis. 46 Suppl 2:S96-100.-   [15] Azad et al. (2013) BMJ. 347:f6471.-   [16] Masco et al. (2003) Systematic and Applied Microbiology,    26:557-563.-   [17] Srútková et al. (2011) J. Microbiol. Methods, 87(1):10-6.-   [18] Ye et al. (2015) PLoS One. 10(1):e0117704.-   [19] Fabro et al. (2015) Immunobiology. 220(1):124-35.-   [20] Yin et al. (2014) Immunogenetics. 66(3):215-8.-   [21] Cheluvappa et al. (2014) Clin Exp Immunol. 175(2):316-22.-   [22] Schieck et al. (2014) J Allergy Clin Immunol. 133(3):888-91.-   [23] Balato et al. (2014) J Eur Acad Dermatol Venerel.    28(8):1016-24.-   [24] Monteleone et al. (2011) BMC Medicine. 2011, 9:122.-   [25] Fahy (2009) Proc Am Thorac Soc 6.256-259-   [26] Miossec and Kolls (2012) Nat Rev Drug Discov. 11(10):763-76.-   [27] Yang et al. (2014) Trends Pharmacol Sci. 35(10):493-500.-   [28] Koenders et al. (2006) J. Immunol. 176:6262-6269.-   [29] Amedei et al. (2012) Int J Mol Sci. 13(10):13438-60.-   [30] Shabgah et al. (2014) Postepy. Dermatol. Alergol. 31(4):256-61.-   [31] Zhang (2015) Inflammation. August 23.-   [32] Sun et al. (2015) Cytokine. 74(1):76-80.-   [33] Mucientes et al. (2015) Br J Ophthalmol. 99(4):566-70.-   [34] Jawad et al. (2013) Ocul Immunol Inflamm. 21(6):434-9.-   [35] Maya et al. (2014) J. Ophthalmology. 310329-   [36] Chi et al. (2007) J. Allergy and Clinical Immunology.    119(5):1218-1224.-   [37] Chi et al. (2008) Investigative Ophthalmology & Visual Science.    49(7): 3058-3064.-   [38] Luger and Caspi (2008) Semin. Immunopathol. 30(2): 134-143.-   [39] Numasaki et al. (2003) Blood. 101:2620-2627.-   [40] Zhang et al. (2008) Biochem. Biophys. Res. Commun. 374:    533-537.-   [41] Karin (2006) Nature. 441: 431-436.-   [42] Faghih et al. (2013). Iranian Journal of Immunology.    10(4):193-204.-   [43] Numasaki et al. (2005) J. Immunol. 175: 6177-6189-   [44] Hammerich and Tacke (2014) Clin Exp Gastroenterol. 7:297-306.-   [45] Haabeth et al. (2012) OncoImmunology 1(1):1146-1152.-   [46] Lejeune et al. (2006) Cancer Immun. 6:6-   [47] Pace et al. (1983) PNAS. 80:8782-6.-   [48] Sgadari et al. (1996) PNAS. 93:13791-6.-   [49] Arenberg et al. (1996) J. Exp. Med. 184:981-92.-   [50] Sgadari et al. (1997) Blood. 89:2635-43.-   [51] Miyamoto-Shinohara et al. (2008) J. Gen. Appl. Microbiol., 54,    9-24.-   [52] Cryopreservation and Freeze-Drying Protocols, ed. by Day and    McLellan, Humana Press.-   [53] Leslie et al. (1995) Appl. Environ. Microbiol. 61, 3592-3597.-   [54] Mitropoulou et al. (2013) J Nutr Metab. (2013) 716861.-   [55] Kailasapathy et al. (2002) Curr Issues Intest Microbiol.    3(2):39-48.-   [56] Handbook of Pharmaceutical Excipients, 2nd Edition, (1994),    Edited by A Wade and P J Weller-   [57] Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R.    Gennaro edit. 1985)-   [58] Handbook of Microbiological Media, Fourth Edition (2010) Ronald    Atlas, CRC Press.-   [59] Maintaining Cultures for Biotechnology and Industry (1996)    Jennie C. Hunter-Cevera, Academic Press-   [60] Strobel (2009) Methods Mol Biol. 581:247-61.-   [61] Gennaro (2000) Remington: The Science and Practice of Pharmacy.    20th edition, ISBN: 0683306472.-   [62] Molecular Biology Techniques: An Intensive Laboratory Course,    (Ream et al., eds., 1998, Academic Press).-   [63] Methods In Enzymology (S. Colowick and N. Kaplan, eds.,    Academic Press, Inc.)-   [64] Handbook of Experimental Immunology, Vols. I-IV (D. M. Weir    and CC. Blackwell, eds, 1986, Blackwell Scientific Publications)-   [65] Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual,    3rd edition (Cold Spring Harbor Laboratory-   Press).-   [66] Handbook of Surface and Colloidal Chemistry (Birdi, K. S. ed.,    CRC Press, 1997)-   [67] Ausubel et al. (eds) (2002) Short protocols in molecular    biology, 5th edition (Current Protocols).-   [68] PCR (Introduction to Biotechniques Series), 2nd ed. (Newton &    Graham eds., 1997, Springer Verlag)-   [69] Current Protocols in Molecular Biology (F. M. Ausubel et al.,    eds., 1987) Supplement 30-   [70] Smith & Waterman (1981) Adv. Appl. Math. 2: 482-489.-   [71] Brand et al. (2007) Nature Protocols. 2(5):1269-1275-   [72] Jiao et al. (2014) Immunopathology and Infectious Diseases.    184(4):1085-93.-   [73] Rockwell et al., (1972) J Natl Cancer Inst. 49:735-49.-   [74] Bertram and Janik (1980) Cancer Lett. 11:63-73.-   [75] Darlington (1987) Meth Enzymol. 151:19-38.-   [76] Principe d'éthique de l'expérimentation animale, Directive no    2010/63 CEE 22 Sep. 2010, Décrét no 2013-118 1 Feb. 2013.-   [77] Guide for the Care and Use of Laboratory Animals: Eighth    Edition. The National Academies Press; 2011-   [78] Simpson-Herren and Lloyd (1970) Cancer Chemother Rep.    54:143-74.-   [79] Workman et al. (2010) Br. J. Cancer. 102:1555-77.-   [80] Bottacini et al (2014) BMC Genomics. 15:170, DOI:    10.1186/1471-2164-15-170, PMID: 24581150

The invention claimed is:
 1. A pharmaceutical composition in the form ofa tablet, capsule, or microcapsule that comprises at least 1×10³ colonyforming units (CFU) per gram of a dried bacteria strain, with respect toa total weight of the pharmaceutical composition and a pharmaceuticallyacceptable excipient, diluent, or carrier; wherein the bacteria straincomprises a biochemical profile that is: (a) positive for raffinosefermentation; (b) positive for at least three of: mannose fermentation,β-galactosidase, arginine arylamidase, proline arylamidase,phenylalanine arylamidase, leucine arylamidase, tyrosine arylamidase,glycine arylamidase, and histidine arylamidase; (c) intermediate for atleast one of: α-galactosidase, α-glucosidase, β-glucosidase, andα-arabinose; and (d) negative for at least five of: urease, argininedihydrolase, β-galactosidase-6-phosphate, β-glucoronidase,N-acetyl-β-glucosaminidase, glutamic acid decarboxylase, α-fucosidase,nitrate reduction, indole production, alkaline phosphatase, leucylglycine arylamidase, pyroglutamic acid arylamidase, alanine arylamidase,glutamyl glutamic acid arylamidase, and serine arylamidase; asdetermined by an Analytical Profile Index test comprising: (i)contacting a suspension of the bacteria strain with a Rapid ID 32A stripat 37° C for at least 4 hours; (ii) contacting the suspension with adevelopment reagent comprising FastBlue for at least 5 minutes; and(iii) measuring a color of the suspension.
 2. The pharmaceuticalcomposition of claim 1, wherein the bacteria strain comprises abiochemical profile that is: (a) positive for raffinose fermentation;(b) positive for arginine arylamidase, proline arylamidase, andphenylalanine arylamidase; (c) intermediate for α-galactosidase; and (d)negative for β-galactosidase-6-phosphate, β-glucoronidase,N-acetyl-β-glucosaminidase, nitrate reduction, and serine arylamidase.3. The pharmaceutical composition of claim 1, wherein the bacteriastrain comprises a biochemical profile that is: (a) positive forraffinose fermentation; (b) positive for at least five of: mannosefermentation, β-galactosidase, arginine arylamidase, prolinearylamidase, phenylalanine arylamidase, leucine arylamidase, tyrosinearylamidase, glycine arylamidase, and histidine arylamidase; (c)intermediate for α-galactosidase; and (d) negative for at least ten of:urease, arginine dihydrolase, β-galactosidase-6-phosphate,glucoronidase, N-acetyl-β-glucosaminidase, glutamic acid decarboxylase,α-fucosidase, nitrate reduction, indole production, alkalinephosphatase, leucyl glycine arylamidase, pyroglutamic acid arylamidase,alanine arylamidase, glutamyl glutamic acid arylamidase, and serinearylamidase.
 4. The pharmaceutical composition of claim 1, wherein thebacteria strain is a butyrate-producing bacteria strain.
 5. Thepharmaceutical composition of claim 1, wherein the pharmaceuticalcomposition is in the form of a capsule.
 6. The pharmaceuticalcomposition of claim 1, wherein a bacterial cell of the bacteria strainbinds to a human cell to a lesser extent than a bacterial cell ofBifidobacterium breve strain JCM 7017 binds to a corresponding humancell, as determined by an in vitro assay comprising comparing ameasurement of an optical density.
 7. The pharmaceutical composition ofclaim 1, wherein the pharmaceutical composition comprises from about1×10⁶ to about 1×10¹¹ CFU per gram, with respect to the total weight ofthe pharmaceutical composition.
 8. The pharmaceutical composition ofclaim 1, further comprising a prebiotic compound.
 9. The pharmaceuticalcomposition of claim 8, wherein the prebiotic compound is selected fromthe group consisting of: fructo-oligosaccharides (or FOS), short-chainfructo-oligosaccharides, inulin, isomalt-oligosaccharides, pectins,xylo-oligosaccharides (or XOS), chitosan-oligosaccharides (or COS),beta-glucans, arable gum modified and resistant starches, polydextrose,D-tagatose, acacia fibers, carob, oats, and citrus fiber.
 10. Thepharmaceutical composition of claim 1, comprising the pharmaceuticallyacceptable carrier, wherein the pharmaceutically acceptable carrier isselected from the group consisting of: lactose, starch, glucose, methylcellulose, magnesium stearate, mannitol and sorbitol.
 11. Thepharmaceutical composition of claim 1, further comprising anantioxidant.
 12. The pharmaceutical composition of claim 11, wherein theantioxidant is ascorbic acid.
 13. The pharmaceutical composition ofclaim 1, wherein the bacteria strain is an anaerobic bacteria strain.14. The pharmaceutical composition of claim 1, wherein the bacteriastrain is a live bacteria strain.
 15. The pharmaceutical composition ofclaim 1, further comprising an anticancer agent.
 16. The pharmaceuticalcomposition of claim 15, wherein the anticancer agent is selected fromthe group consisting of: an immune checkpoint inhibitor, a targetedantibody immunotherapy, a CAR-T cell therapy, an oncolytic virus, and acytostatic drug.
 17. The pharmaceutical composition of claim 1, whereinthe bacteria strain is positive for methyl α-D-glucopyranosidefermentation, as determined by an API 50 CH analytical profile test. 18.The pharmaceutical composition of claim 1, wherein the bacteria strainis positive for methyl D-melibiose fermentation, as determined by an API50 CH analytical profile test.